Patent application title: SCREENING ASSAYS TO IDENTIFY COMPOUNDS WHICH MODULATE T1R ASSOCIATED TASTE MODALITIES WHICH ELIMINATE FALSE POSITIVES
Inventors:
Guy Servant (San Diego, CA, US)
Nicole Servant (San Diego, CA, US)
IPC8 Class: AG01N33566FI
USPC Class:
1 1
Class name:
Publication date: 2019-09-12
Patent application number: 20190277845
Abstract:
This invention relates to assays which screen for compounds that modulate
taste elicited by the T1R2/T1R3 sweet taste receptor which include a
novel counter screen to eliminate false positives. In addition, the
invention contemplates assays which screen for compounds that modulate
taste elicited by the T1R1/T1R3 umami taste receptor which include a
novel counter screen to eliminate false positives. Preferably the assays
are conducted in high throughput format thereby enabling the screening of
many hundreds of different compounds whereby the counter screen
significantly improves assay efficiency. Further, the invention relates
to the use of the compounds identified in the subject screening assays to
modulate T1R associated taste perception.Claims:
1-27. (canceled)
28. A method of screening for a compound that putatively modulates umami taste wherein such method reduces or eliminates "false positives", the method comprising the steps of: (i) screening one or more compounds in an assay that identifies compounds which specifically bind or modulate the activity of a T1R1/T1R3 heteromeric taste receptor or which modulate the binding or the activation of a T1R1/T1R3 heteromeric taste receptor by another compound; (ii) further conducting a counter-screen using one or more of the identified putative T1R1/T1R3 modulatory compounds identified n step (i), wherein said counter-screen detects whether said one or more identified putative T1R1/T1R3 modulatory compounds specifically bind or modulate the activity of a homomeric T1R1 polypeptide or whether said one or more identified putative T1R1/T1R3 modulatory compounds modulate the binding or the activation of a homomeric T1R1 taste receptor polypeptide by another T1R1 agonist compound; and (iii) based on the results of the counter-screen identifying whether the identified compound or compounds are "false positives" or are compounds that putatively modulate umami taste.
29-59. (canceled)
60. A high throughput screening method for identifying compounds that putatively modulate sweet taste wherein such method reduces or eliminates "false positives", the method comprising the steps of: (i) screening a thousand or more compounds in an assay that identifies compounds which specifically bind to or modulate the activity of a human T1R2/T1R3 heteromeric taste receptor or which modulate the binding or the activation of a human T1R2/T1R3 heteromeric taste receptor by another compound; (ii) further conducting a counter-screen using the identified putative human T1R2/T1R3 modulatory compounds identified from the thousand or more compounds screened in step (i), wherein said counter-screen detects whether said one or more identified putative human T1R2/T1R3 modulatory compounds specifically bind to or modulate the activity of a homomeric human T1R2 polypeptide or whether said one or more identified putative human T1R2/T1R3 modulatory compounds modulate the binding or the activation of a homomeric human T1R2 polypeptide by another human T1R2 agonist compound; and (iii) assessing the effect of the compounds which test positive in (i) and not in (ii) in human taste tests which assay the effect of said compounds on sweet taste, wherein said screening method does not include any other binding or functional assays using T1R taste receptor polypeptides and further does not screen using a cell which endogenously expresses said human T1R2/T1R3 taste receptor or a cell which endogenously expresses said homomeric human T1R2 polypeptide.
61. The method of claim 60 which further comprises and producing variants or derivatives of the compounds that are identified as eliciting or modulating sweet taste in said human taste tests and identifying those variants and derivatives which are suitable for use as flavorants in foods, beverages, medicaments or comestibles for human or animal consumption in the development of variants or derivatives.
62. The method of claim 60, wherein step (i) of the method uses eukaryotic cells which stably or transiently express human T1R2/T1R3 and the counter-screen uses cells that stably or transiently express a human T1R2 polypeptide and which do not express any T1R3 polypeptide.
63. The method of claim 60, wherein step (i) uses a eukaryotic cell membrane comprising said human T1R2/T1R3 receptor.
64. The method of claim 60, wherein said human T1R2/T1R3 receptor in step (i) and said human T1R2 homomeric receptor are both expressed by a eukaryotic cell.
65. The method of claim 64, wherein said cell is a yeast, fungus, insect, oocyte, worm, or mammalian cell.
66. The method of claim 65, wherein the mammalian cell is a human or rodent cell.
67. The method of claim 64, wherein said eukaryotic cell is a CHO, COS, U2OS, NIH3T3 or MDCK cell, BHK, HeLa, or STO cell or comprises a Xenopus oocyte.
68. The method of claim 60, wherein the human T1R2/T1R3 heteromer and/or the homomeric human T1R2 receptor is linked to a solid phase.
69. The method of claim 66, wherein said eukaryotic cell further expresses a G protein that couples to said human T1R2/T1R3 heteromer and the homomeric human T1R2 polypeptide.
70. The method of claim 69, wherein said G protein is G.alpha.15, G.alpha.16, transducin, gustducin or a chimera of any of the foregoing G proteins.
71. The method of claim 70, wherein said G protein is a chimera of G.alpha.15 or G.alpha.16, and gustducin.
72. The method of claim 70, wherein said G protein is a chimera of G.alpha.15 or G.alpha.16, and transducin.
73. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric human T1R2 polypeptide are measured by detecting changes in intracellular Ca2+ levels.
74. The method of claim 73, wherein Ca2+ levels are detected using an ion sensitive dye or a membrane voltage fluorescent indicator.
75. The method of claim 60, which uses a FLIPR detection system.
76. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric human T1R2 polypeptide are measured by monitoring changes in fluorescence polarization.
77. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric human T1R2 polypeptide are measured by detecting changes in second messenger levels.
78. The method of claim 77, wherein said second messenger is IP3.
79. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric human T1R2 polypeptide are measured by detecting changes in intracellular cyclic nucleotides.
80. The method of claim 79, wherein said cyclic nucleotide is cAMP or cGMP.
81. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric T1R2 polypeptide are measured by measuring changes is Ca2+ levels by fluorescence imaging.
82. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric human T1R2 polypeptide are measured by detecting changes in G protein binding of GTP.delta.S.
83. The method of claim 60, wherein the human T1R2 polypeptide is at least 90% identical to a T1R2 polypeptide having or encoded by a sequence selected from SEQ ID NO:3, 7, 13, and 14.
84. The method of claim 60, wherein the human T1R2 polypeptide is at least 95% identical to a human T1R2 polypeptide having or encoded by a sequence selected from SEQ ID NO:3, 7, 13, and 14.
85. The method claim 60, wherein the human T1R3 polypeptide is at least 90% identical to a human T1R3 polypeptide having or encoded by a sequence selected from SEQ ID NO:1, 4, 6, and 8.
86. The method claim 60, wherein the human T1R3 polypeptide is at least 95% identical to a human T1R3 polypeptide having or encoded by a sequence selected from SEQ ID NO: 1, 4, 6, and 8.
Description:
[0001] This application claims priority to U.S. Ser. No. 61/789,993 filed
Mar. 15, 2013. The contents of said application are incorporated by
reference in its entirety herein.
FIELD OF THE INVENTION
[0002] This invention relates to assays which screen for compounds that modulate the T1R2/T1R3 sweet taste receptor which include a novel counter screen to eliminate false positives. In addition, the invention contemplates assays which screen for compounds that modulate the T1R1/T1R3 umami taste receptor which include a novel counter screen to eliminate false positives. Preferably the assays are conducted in high throughput format thereby enabling the screening of many hundreds of different compounds whereby the counter screen significantly improves assay efficiency. Further, the invention relates to the use of the compounds identified in the subject screening assays to modulate T1R associated taste perception.
BACKGROUND OF THE INVENTION
[0003] Researchers affiliated with the Assignee Senomyx as well as a research group at the University of California have previously reported the identification and functionalization of a family of G-protein coupled receptors involved in mammalian taste referred to as the T1R family. This family of taste receptors consists of 3 members, T1R1, T1R2 and T1R3. These receptors are expressed in different tissues including the tongue, organs in the digestive system as well as in other types of tissues and modulate sweet and umami taste.
[0004] In particular, these entities have demonstrated using mammalian cells that express the T1R1 and T1R3 as well as a suitable G protein, e.g., a promiscuous G protein or a chimeric G protein, that the T1R1 and T1R3 receptors dimerize to form a heteromeric taste receptor comprising T1R1 and T1R3 polypeptides that responds to umami (savory) taste compounds. These same entities have similarly demonstrated that when the T1R2 and T1R3 polypeptides are expressed in a cell that comprises a suitable G protein, e.g., a promiscuous G protein or a chimeric G protein, that the T1R2 and T1R3 polypeptides dimerize to form a heteromeric taste receptor comprising the T1R2 and T1R3 polypeptides that responds to sweet taste compounds. This research is reported in various scientific articles including Li et al., Proc. Natl. Acad. Sci, USA 2002, April 12 99(7)4692-4696; Xu et al., Proc. Natl. Acad. Sci, USA 2004, September 28, 101(36)14652863; as well as numerous patents assigned to Senomyx and the University of California.
[0005] Also, these entities and others have disclosed the use of recombinant and endogenous cells which co-express either T1R2 and T1R3 polypeptides or T1R1 and T1R3 polypeptides to respectively identify compounds that modulate sweet or umami taste. Also, T1R polypeptides have been suggested to be involved in glucose transport, food sensing and motility, and insulin responses.
[0006] Assays using T1R2/T1R3 and T1R1/T1R3 heteromers have resulted in the identification of numerous compounds that modulate sweet or umami taste some of which have been approved for use in foods for human consumption. While these assays are predictive as to the potential effect of the identified compounds on a particular taste modality associated with T1R-associated taste, i.e., sweet or umami taste, one recurring problem which has been observed is that the "hits", i.e., the group of identified T1R2/T1R3 or T1R1/T1R3 modulatory compounds often contain "false positives". That is to say, while the compounds demonstrably modulate the activity of the sweet or umami heteromeric taste receptor in vitro, they do not elicit a demonstrable or desired effect on sweet or umami taste in humans.
[0007] One way of potentially alleviating such "false positives" is the use of cells that endogenously express the T1R2/T1R3 or T1R1/T1R3 receptors rather than recombinant cells as these cells may be less susceptible to interacting with non-physiologically relevant compounds. The present invention provides another means for eliminating such "false positives" in screening assays using the T1R2/T1R3 sweet receptor which has been demonstrated with numerous compounds to be highly reliable.
BRIEF DESCRIPTION AND OBJECTS OF THE INVENTION
[0008] It is an object of the invention to improve the efficiency of screening assays for identifying compounds that modulate sweet taste in humans and potentially other animals such as rodents, dogs, cats, and other animals used in agriculture.
[0009] More specifically, is an object of the invention to provide improved screening assays for identifying compounds that modulate sweet taste in humans and potentially other animals such as rodents, dogs, cats, and other animals used in agriculture, wherein the improvement substantially reduces the number of "false positives".
[0010] More specifically, it is an object of the invention to conduct screening assays to identify compounds that modulate sweet taste wherein the assay comprises (i) conducting a binding or functional assay to identify compounds that bind to or modulate the activity of the T1R2/T1R3 receptor, (ii) further conducting a counter-screen to assess whether the positive "hits" in step (i) bind to and/or modulate the activity of homomeric T1R2; (iii) if said compounds bind to and/or modulate the activity of homomeric T1R2, identifying such compounds as likely "false positives" which will not elicit a desired effect on sweet taste.
[0011] It is also an object of the invention to conduct screening assays to identify compounds that modulate umami taste wherein the assay comprises (i) conducting a binding or functional assay to identify compounds that bind to or modulate the activity of the T1R1/T1R3 receptor, (ii) further conducting a counter-screen to assess whether the positive "hits" in step (i) bind to and/or modulate the activity of homomeric T1R1; (iii) if said compounds bind to and/or modulate the activity of homomeric T1R1, identifying such compounds as likely "false positives" which will not elicit a desired effect on umami taste.
[0012] It is also an object of the invention to use the compounds identified by the present screening assays for the development of flavorants for use in different foods, beverages, medicaments or comestibles for human or animal consumption, as well as to synthesize optimized variants or derivatives of these compounds for use in different foods, beverages, medicaments or comestibles for human or animal consumption.
SUMMARY OF THE INVENTION
[0013] The present invention provides a novel use of hT1R2 to identify compounds which while activating the T1R2/T1R3 taste receptor in in vitro screening assays, may exhibit poor effects in human taste tests. Particularly, the present invention provides the use of homomeric T1R2 in screening assays to eliminate or reduce the number of false positives identified in screening assays using heteromeric T1R2/T1R3 taste receptors.
[0014] Also, the present invention provides a novel use of hT1R1 to identify compounds which while activating the T1R1/T1R3 taste receptor in in vitro screening assays, may exhibit poor effects in human taste tests. Particularly, the present invention contemplates the use of homomeric T1R1 in screening assays to eliminate or reduce the number of false positives identified in screening assays using heteromeric T1R1/T1R3 taste receptors.
[0015] As noted above, the human sweet taste receptor requires two subunits, named T1R2 and T1R3, to function and be activated by known sweeteners. Recently, it was allegedly reported in U.S. Pat. No. 8,124,360, by Slack, assigned to Givaudan, that homomeric T1R2 (defined as a monomer, dimer or oligomer of the T1R2 polypeptide) functionally responds to Perillartine, a synthetic oxime that is approximately 370-times more potent/sweet than sucrose). The reference does not contain any data indicating other sweet compounds activate T1R2. Based on their purported results with Perillartine, the patent suggests that T1R2 monomers activated by Perillartine may be used in direct functional screens to identify compounds that modulate sweet taste signaling and which modulate sweet taste in humans.
[0016] The present inventors recently screened cells that express T1R2 and which do not express T1R3 and discovered several compounds that effectively activate the hT1R2 subunit when expressed in the absence of T1R3 in heterologous cells. Based on this activation, these compounds were evaluated in human taste tests. As disclosed infra in the working examples, when tested the identified compounds which activated hT1R2 expressed in the absence of T1R3 behaved poorly in human taste tests. Particularly, they were much less potent then what would have been predicted from their potency measured with cells expressing the sweet taste receptor hT1R2/hT1R3.
[0017] Based on these results, the inventors hypothesized that cells expressing hT1R2 and hT1R3 likely express at least two types of functional receptors, most likely both dimers, but potentially an active monomer or other heteromer or oligomer. Whereas the heterodimer hT1R2/hT1R3 responds to compounds that are physiologically relevant, it was theorized that the homodimer form of T1R2 (i.e., hT1R2/hT1R2 or potentially a T1R2 monomer or oligomer or heteromer comprising T1R2 subunits) responds to compounds that are not physiologically relevant and based thereon will not elicit a favorable response in human or other animal taste tests. Based on these hypotheses it was further theorized that an hT1R2 homomeric assay, instead of purportedly being useful to identify sweet taste modulatory compounds, instead may be useful in identifying compounds, discovered using an hT1R2/hT1R3 assay that will ultimately behave poorly in human taste tests. That is to say the assay using the homomeric form of T1R2 will function as a counter-screen to identify "false positives" in the T1R2/T1R3 screening assays, i.e., compounds which apparently modulate T1R2/T1R3 in in vitro screening assays, but which elicit poor properties in taste tests.
[0018] In fact, the inventors' hypotheses were confirmed. As taught in Examples 1-7 infra, compounds identified as agonists in a fluorometric cell-based hT1R2/hT1R3 assay which detects receptor activity by detecting levels of intracellular calcium were screened using the same fluorometric cell-based assays, but instead using cells that only express T1R2. Seventeen compounds or sweeteners which had been identified as agonists in the hT1R2/hT1R3 assay and showing a good correlation between taste data and assay data (within a 95% confidence interval) were inactive in the hT1R2 assay. Conversely, six compounds identified as agonists in the hT1R2/hT1R3 assay were found to behave poorly in human taste tests or to be tasteless were active in the hT1R2 assay. Based on these results homomeric T1R2 assays may be used in screening assays as an adjunct to T1R2/T1R3 screening assays as a counter-screen, i.e., to eliminate false positives, e.g., compounds which are active in the T1R2/T1R3 screens, but which elicit poor results in taste tests.
[0019] Based thereon, the present invention is preferably directed to improve T1R2/T1R3 modulator screening assays, preferably cell-based functional or binding T1R2/T1R3 screening assays, wherein the improvement includes use of a counter-screen that eliminates false positives. The counter screen comprises conducting cell-based screening assays with compounds identified to modulate T1R2/T1R3 using cells that express T1R2 in the absence of T1R3. Preferably these assays are fluorometric assays using the FLIPR system. However, it is contemplated that other T1R binding assays or functional assays used to identify potential T1R modulators may be used. Such assays are disclosed herein. The use of this counter-screen should minimize false positives based on their binding or modulation of the activity of the homomeric T1R2 and thereby facilitate the discovery and development of compounds that modulate sweet taste.
[0020] In addition, the present invention encompasses improved T1R1/T1R3 modulator screening assays, preferably cell-based functional or binding assays screening for T1R1/T1R3 modulators, wherein the improvement includes use of a counter-screen that eliminates false positives. Similarly, the T1R1 counter screen will comprise conducting cell-based screening assays with compounds identified to modulate T1R1/T1R3 using cells that express T1R1 in the absence of T1R3. Preferably these assays are fluorometric assays using the FLIPR system. Such assays are disclosed herein. However, it is contemplated that other T1R binding assays or functional assays used to identify potential T1R1/T1R3 modulators. Similarly, this counter-screen should minimize false positives based on their binding or modulation of the activity of the homomeric T1R1 and thereby facilitate the discovery and development of compounds that modulate umami taste.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound A, a sweet taste receptor agonist known to bind to the transmembrane domain of hT1R2.
[0022] FIG. 2 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound B, a sweet taste receptor agonist.
[0023] FIG. 3 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound C, a sweet taste receptor agonist.
[0024] FIG. 4 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound D, a sweet taste receptor agonist.
[0025] FIG. 5 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by Perillartine, a sweet taste receptor agonist.
[0026] FIG. 6 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound P-4000, a sweet taste receptor agonist.
[0027] FIG. 7 contains potency data at different molar ratios in comparison to sucrose for 17 compounds or sweeteners identified as agonists in the hT1R2/hT1R3 assay in comparison to their relative sweetness to sucrose in human sweet taste tests.
DETAILED DESCRIPTION OF THE INVENTION
[0028] As disclosed supra, the present invention describes a novel use of hT1R2 to identify compounds which while activating the T1R2/T1R3 taste receptor in in vitro screening assays, exhibit poor effects in human taste tests. Particularly, the present invention provides the use of homomeric T1R2 in screening assays to eliminate or reduce the number of false positives identified in screening assays using heteromeric T1R2/T1R3 taste receptors.
[0029] Also, based on the results with T1R2, the present invention further contemplates a novel use of hT1R1 to identify compounds which while activating the T1R1/T1R3 taste receptor in in vitro screening assays, exhibit poor effects in human taste tests. Particularly, the present invention provides the use of homomeric T1R1 in screening assays to eliminate or reduce the number of false positives identified in screening assays using heteromeric T1R1/T1R3 taste receptors.
[0030] As noted above, the human sweet taste receptor requires two subunits, named T1R2 and T1R3, to function and be activated by known sweeteners. Likewise, the human umami taste receptor requires two subunits, named T1R1 and T1R3, to function and be activated by known umami compounds. Recently, it was reported in U.S. Pat. No. 8,124,360, by Slack, and assigned to Givaudan that homomeric T1R2 (defined as a monomer, dimer or oligomer of the T1R2 polypeptide) functionally responds to Perillartine, a synthetic oxime that is approximately 370-times more potent/sweet than sucrose). The reference does not contain any data indicating other sweet compounds activate T1R2. However, based on their purported results with Perillartine, the patent suggests that T1R2 monomers activated by Perillartine may be used in direct functional screens to identify compounds that modulate sweet taste signaling and which modulate sweet taste in humans. Also, it has been reported that homomeric T1R3 binds to some sweet compounds.
[0031] The present inventors recently screened cells that express T1R2 and which do not express T1R3 and discovered several compounds that effectively activate the hT1R2 subunit when expressed on its own in heterologous cells. Based on this activation, these compounds were evaluated in human taste tests. It was initially predicted that such compounds might elicit sweet taste. However, when tested the identified compounds which activated hT1R2 expressed in the absence of T1R3 behaved poorly in human taste tests, being much less potent then what would have been expected from their potency measured with cells expressing the sweet taste receptor hT1R2/hT1R3.
[0032] Based on these results, the inventors hypothesized that cells expressing hT1R2 and hT1R3 likely express at least two types of functional receptors, most likely both dimers, however potentially an active monomer, oligomer or other heteromer. It was predicted that whereas the heterodimer hT1R2/hT1R3 responds to compounds that are physiologically relevant; that the homodimer form of T1R2 (i.e., hT1R2/hT1R2 or potentially a T1R2 monomer or oligomer comprising T1R2 subunits) responds to compounds that are not physiologically relevant and based thereon will not elicit a favorable response in human or other animal taste tests.
[0033] Based on these hypotheses it was further theorized that an hT1R2 homomeric assay, instead of being useful to identify sweet taste modulatory compounds, instead may serve an opposite purpose. Particularly, the homomeric T1R2 may be useful in identifying compounds, discovered using an hT1R2/hT1R3 functional or binding assay, and that will ultimately behave poorly in human taste tests. That is to say the assay using the homomeric form of T1R2 will function as a counter-screen to identify "false positives" in the T1R2/T1R3 screening assays, i.e., compounds which apparently modulate T1R2/T1R3 in in vitro screening assays, but which elicit poor properties in taste tests.
[0034] As substantiated by the examples, the inventors' hypotheses were confirmed. Compounds identified as agonists in a fluorometric cell-based hT1R2/hT1R3 assay which detects receptor activity by detecting levels of intracellular calcium were screened using the same fluorometric cell-based assays, but instead using cells that only express T1R2. Seventeen compounds or sweeteners identified as agonists in the hT1R2/hT1R3 assay and showing a good correlation between taste data and assay data (within a 95% confidence interval) were inactive in the hT1R2 assay. Conversely, six compounds identified as agonists in the hT1R2/hT1R3 assay were found to behave poorly in human taste tests or to be tasteless were active in the hT1R2 assay. Based on these results homomeric T1R2 assays may be used in screening assays as an adjunct to T1R2/T1R3 screening assays as a counter-screen, i.e., to eliminate false positives, e.g., compounds which are active in the T1R2/T1R3 screens, but which elicit poor results in taste tests.
[0035] Based thereon, the present invention is preferably directed to improve T1R2/T1R3 modulator screening assays, preferably cell-based functional or binding T1R2/T1R3 screening assays, wherein the improvement includes use of a counter-screen that eliminates false positives. The counter screen comprises conducting screening assays, preferably cell-based, with compounds identified to modulate activity in T1R2/T1R3 screening assays, using T1R2 in the absence of T1R3. Preferably these assays are cell-based fluorimetric assays using the FLIPR system. However, it is contemplated that other T1R binding assays or functional assays used to identify potential T1R modulators may be used. Suitable examples of such assays are disclosed herein and are known in the art. The use of this T1R2 counter-screen should minimize false positives based on their binding or modulation of the activity of the homomeric T1R2 and thereby facilitate the discovery and development of compounds that modulate sweet taste.
[0036] In addition, the present invention encompasses improved T1R1/T1R3 modulator screening assays, preferably cell-based functional or binding assays screening for T1R1/T1R3 modulators, wherein the improvement includes use of a counter-screen that eliminates false positives. Similarly, the counter screen will comprise conducting cell-based screening assays with compounds identified to modulate activity in T1R1/T1R3 screening assays, using T1R1 in the absence of T1R3. Preferably these assays are cell-based fluorimetric assays using the FLIPR system. Such assays are disclosed herein. However, it is contemplated that other T1R binding assays or functional assays used to identify potential T1R1/T1R3 modulators. Similarly, this counter-screen should minimize false positives based on their binding or modulation of the activity of the homomeric T1R1 and thereby facilitate the discovery and development of compounds that modulate umami taste.
[0037] More specifically, the present invention also provides assays, preferably high throughput assays, which include a counter screen to identify molecules that interact with and/or modulate T1R2/T1R3 or T1R1/T1R3 polypeptide hetero-oligomeric complexes. These assays typically may use intact T1R polypeptides and heteromers containing. However, it is further contemplated that the initial heteromer screening assays and potentially the counter screen assays may be conducted with monomers or heteromeric T1Rs comprised of chimeric T1R subunits, i.e., comprising the extracellular domain of a T1R and the transmembrane region of another GPCR such as the calcium sensing receptor, or potentially another species T1R or the extracellular domain of a particular GPCR, potentially another species T1R or that of another GPCR such as the calcium sensing receptor and the transmembrane region of a T1R. It has been shown that heteromers comprised of T1R2/T1R3 heteromers and T1R1/T1R3 heteromers comprised of chimeric T1R2/T1R3 subunits or chimeric T1R1/T1R3 subunits are functional and may be used to isolate ligands, agonists, antagonists, or any other molecules that can bind to and/or modulate the activity of a T1R polypeptide. Also, the present invention contemplates assays, preferably high throughput assays, which include a counter screen to identify molecules that interact with and/or modulate T1R2/T1R3 or T1R1/T1R3 polypeptide hetero-oligomeric complexes, wherein the T1R subunits may comprise variants or fragments of a particular T1R, preferably human or rodent T1R2, T1R3 or T1R1, which are functional (bind or functionally respond to sweet or umami compounds). These T1R variants include T1R1, T1R2 and T1R3 polypeptides that are at least 80, 90, 95, 96, 97, 98, or at least 99% identical to particular native T1R sequences including those disclosed in the Sequence Listing and known in the art. For example. The nucleic acid and polypeptide sequences of different species T1R1, T1R2 and T1R3 including human, mouse, rat, cat and dog are in the public domain.
Definitions
[0038] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
[0039] "Taste cells" herein include any cell, which taste cell may be an endogenous T1R expressing cell or may comprise a recombinant cell that expresses at least one T1R1, T1R2 or T1R3 subunit, preferably human or rodent T1R subunits and typically a G protein that functionally couples to the T1R subunit or T1R subunits expressed in the taste cell. This includes by way of example neuroepithelial cells that are organized into groups to form taste buds of the tongue, e.g., foliate, fungiform, and circumvallate cells (see, e.g., Roper et al., Ann. Rev. Neurosci. 12:329-353 (1989)). Taste cells are also found in the palate and other tissues, such as the esophagus and the stomach as well as being expressed in other tissues and organs including by way of example those comprised in other tissues of the gastrointestinal system, the urinary system, the nervous system, the reproductive system, the skin and others.
[0040] "T1R" refers to one or more members of a family of G protein-coupled receptors that are expressed in taste cells such as foliate, fungiform, and circumvallate cells, as well as cells of the palate, and esophagus (see, e.g., Hoon et al., Cell, 96:541-551 (1999), herein incorporated by reference in its entirety). The current nomenclature for the T1R family members is T1R1, T1R2 and T1R3. Members of this family are also referred to as GPCR-B3 and TR1 in WO 00/06592 as well as GPCR-B4 and TR2 in WO 00/06593. T1R1 has also been referred to as GPCR-B3, and T1R2 as GPCR-B4. T1R family members are involved in sweet and umami taste perception.
[0041] "T1R's" are comprised in a family of GPCRs with seven transmembrane regions that have "G protein-coupled receptor activity," e.g., they may bind to G proteins in response to extracellular stimuli and promote production of second messengers such as IP3, cAMP, cGMP, and Ca.sup.2+ via stimulation of enzymes such as phospholipase C and adenylate cyclase (for a description of the structure and function of GPCRs, see, e.g., Fong, supra, and Baldwin, supra). Like some other GPCRs the T1Rs dimerize to produce functional taste receptors, i.e., the T1R1 and T1R3 subunits dimerize to produce the umami taste receptor and the T1R2 and T1R3 subunits dimerize to produce the sweet taste receptor.
[0042] The term "T1R" herein includes polymorphic variants, alleles, mutants, and interspecies homologs of T1R1, T1R2 and T1R3 that: (1) have at least about 70-80% amino acid sequence identity, optionally about 85, 90, 95, 96, 97, 98, or 99% amino acid sequence identity to any of the T1R polypeptide sequences contained in the Sequence Listing or known in the art over a window of about 25 amino acids, optionally 50-100 amino acids, and more typically over at least 200, 300, 400, 500, 600 amino acids, or the entire length of the particular T1R polypeptide or at least the extracellular or transmembrane region thereof; (2) polypeptides which are encoded by a nucleic acid molecule encoding a T1R nucleic acid contained in the Sequence Listing or known in the art; or (iii) a T1R polypeptide encoded by a nucleic acid which specifically hybridizes under stringent hybridization conditions to the complement of any of the T1R nucleic acid sequences contained in the Sequence Listing or known in the art or a fragment thereof which is at least about 100, optionally at least about 200, 300, 400, or at least 500-1000 nucleotides and (3) chimeric polypeptides that comprise a polypeptide which is at least 90% identical to the extracellular region of a particular T1R and the transmembrane region of another GPCR other than said T1R and chimeric polypeptides that comprise a polypeptide which is at least 90% identical to the transmembrane region of a T1R and the extracellular region of another GPCR other than said T1R.
[0043] "Homomeric T1R2" herein refers to a T1R2 polypeptide defined as above, or a chimeric T1R2 polypeptide, that is expressed in the absence of a T1R3 polypeptide or which is in a composition that does not contain a T1R3 polypeptide. This potentially includes T1R2/T1R2 dimers, monomeric T1R2, as well as other oligomers of T1R2 or heteromers of T1R2 not comprising T1R3. Preferably such homomeric T1R2 will functionally respond to Perillartine and/or P-4000. Homomeric T1R2 is preferably obtained by the expression of a T1R2 in a cell that does not express T1R3, and which expresses a G protein such as a promiscuous G protein, e.g., G.alpha.15 or G.alpha.16, or transducin, gustducin, or a chimera of any of these G proteins, preferably a chimera of G.alpha.15 or G.alpha.16 and transducin or gustducin wherein the C-terminal 25-44 amino acids are the same as in gustducin or transducin.
[0044] "Homomeric T1R1" herein refers to a T1R1 polypeptide defined as above, or a chimeric T1R1 polypeptide, that is expressed in the absence of a T1R3 polypeptide or which is in a composition that does not contain a T1R3 polypeptide. This potentially includes T1R1/T1R1 dimers, monomeric T1R1, as well as other oligomers of T1R1 or heteromers of T1R1 not comprising T1R3.
[0045] Topologically, chemosensory GPCRs including T1Rs have an "N-terminal domain;" "extracellular domains;" "transmembrane domains" comprising seven transmembrane regions, and corresponding cytoplasmic, and extracellular loops; "cytoplasmic domains," and a "C-terminal domain" (see, e.g., Hoon et al., Cell, 96:541-551 (1999); Buck & Axel, Cell, 65:175-187 (1991)). These domains can be structurally identified using methods known to those of skill in the art, such as sequence analysis programs that identify hydrophobic and hydrophilic domains (see, e.g., Stryer, Biochemistry, (3rd ed. 1988); see also any of a number of Internet based sequence analysis programs, such as those found at dot.imgen.bcm.tmc.edu). Such domains are useful for making chimeric proteins and for in vitro assays of the invention, e.g., ligand-binding assays.
[0046] "Extracellular domains" therefore refers to the domains of T1R polypeptides that protrude from the cellular membrane and are exposed to the extracellular face of the cell. Such domains generally include the "N terminal domain" that is exposed to the extracellular face of the cell, and optionally can include portions of the extracellular loops of the transmembrane domain that are exposed to the extracellular face of the cell, i.e., the loops between transmembrane regions 2 and 3, between transmembrane regions 4 and 5, and between transmembrane regions 6 and 7.
[0047] The "N-terminal domain" region starts at the N-terminus and extends to a region close to the start of the transmembrane domain. More particularly, in one embodiment of the invention, this domain starts at the N-terminus and ends approximately at the conserved glutamic acid at amino acid position 563 plus or minus approximately 20 amino acids. These extracellular domains are useful for in vitro ligand-binding assays, both soluble and solid phase. In addition, transmembrane regions, described below, can also bind ligand either in combination with the extracellular domain, and are therefore also useful for in vitro ligand-binding assays.
[0048] "Transmembrane domain," which comprises the seven "transmembrane regions," refers to the domain of T1R polypeptides that lies within the plasma membrane, and may also include the corresponding cytoplasmic (intracellular) and extracellular loops. In one embodiment, this region corresponds to the domain of T1R family members which starts approximately at the conserved glutamic acid residue at amino acid position 563 plus or minus 20 amino acids and ends approximately at the conserved tyrosine amino acid residue at position 812 plus or minus approximately 10 amino acids. The seven transmembrane regions and extracellular and cytoplasmic loops can be identified using standard methods, as described in Kyte & Doolittle, J. Mol. Biol., 157:105-32 (1982)), or in Stryer, supra.
[0049] "Cytoplasmic domains" refers to the domains of T1R polypeptides that face the inside of the cell, e.g., the "C-terminal domain" and the intracellular loops of the transmembrane domain, e.g., the intracellular loop between transmembrane regions 1 and 2, the intracellular loop between transmembrane regions 3 and 4, and the intracellular loop between transmembrane regions 5 and 6. "C-terminal domain" refers to the region that spans the end of the last transmembrane domain and the C-terminus of the protein, and which is normally located within the cytoplasm. In one embodiment, this region starts at the conserved tyrosine amino acid residue at position 812 plus or minus approximately 10 amino acids and continues to the C-terminus of the polypeptide.
[0050] The term "ligand-binding region" or "ligand-binding domain" refers to sequences derived from a chemosensory receptor, particularly a taste receptor that substantially incorporates at least the extracellular domain of the receptor. In one embodiment, the extracellular domain of the ligand-binding region may include the N-terminal domain, the transmembrane domain, and portions thereof such as the extracellular loops of the transmembrane domain and in particular amino acids located in the membrane which may bind ligands. The ligand-binding region may be capable of binding a ligand, and more particularly, a tastant.
[0051] The phrase "heteromer" or "hetero-oligomeric complex" in the context of the T1R receptors or polypeptides of the invention refers to a functional combination of at least two T1R receptors or polypeptides, at least one T1R receptor or polypeptide and another taste-cell-specific GPCR, or a combination thereof, to thereby effect chemosensory taste transduction. For instance, the receptors or polypeptides may be co-expressed within the same taste receptor cell type, and the two receptors may physically interact to form a hetero-oligomeric or heteromeric taste receptor.
[0052] The phrase "functional effects" in the context of assays for testing compounds that modulate T1R family member mediated taste transduction includes the determination of any parameter that is indirectly or directly under the influence of the receptor, e.g., functional, physical and chemical effects. It includes ligand binding, changes in ion flux, membrane potential, current flow, transcription, G protein binding, GPCR phosphorylation or dephosphorylation, signal transduction, receptor-ligand interactions, second messenger concentrations (e.g., cAMP, cGMP, IP3, or intracellular Ca.sup.2+), in vitro, in vivo, and ex vivo and also includes other physiologic effects such as increases or decreases of neurotransmitter or hormone release.
[0053] By "determining the functional effect" in the context of assays is meant assays for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a T1R family member, e.g., functional, physical and chemical effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g. fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties, patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, oocyte T1R gene expression; tissue culture cell T1R expression; transcriptional activation of T1R genes; ligand-binding assays; voltage, membrane potential and conductance changes; ion flux assays; changes in intracellular second messengers such as cAMP, cGMP, and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, and the like.
[0054] "Inhibitors," "activators," "enhancers" and "modulators" of T1R proteins include any compound that binds to or which modulates (increases or decreases) the activity of a T1R or T1R heteromer or homomeric T1R or which modulates (increase or decrease) the binding of another compound to a particular T1R monomer or a T1R heterodimer or T1R homodimer. These compounds may be identified in in vitro or in vivo assays, preferably cell-based assays that assay the effect of a compound on the activity of a particular T1R polypeptide or heteromer, typically T1R1/T1R3 or T1R2/T1R3 heteromers, and preferably human T1R1/T1R3 or T1R2/T1R3 heteromers. This includes agonists, antagonists, and their homologs and mimetics. Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate T1R taste transduction, e.g., antagonists. Activators or enhancers are compounds that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize, or up regulate T1R taste transduction, e.g., agonists. T1R modulators can include genetically modified versions of T1R family members, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Assays for identifying T1R inhibitors and activators include, e.g., expressing T1R family members in cells or cell membranes, applying putative modulator compounds, in the presence or absence of tastants, e.g., sweet tastants, and then determining the functional effects on taste transduction, as described above. Samples or assays comprising T1R family members that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of modulation. Control samples (untreated with modulators) are assigned a relative T1R activity value of 100%. Inhibition of a T1R is achieved when the T1R activity value relative to the control is about 80%, optionally 50% or 25-0%. Activation of a T1R is achieved when the T1R activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
[0055] "Counter-screen" herein refers to an assay using a homomeric T1R, preferably homomeric T1R2 or T1R1, more preferably human homomeric T1R2 (T1R2 expressed in the absence of T1R3), that is designed to identify potential "false positives", i.e., compounds which modulate the activity of T1R2/T1R3 in vitro or T1R1/T1R3 in vitro, but which do not elicit a desired effect on taste, e.g., sweet or umami taste. Such counter-screen assay will evaluate the effect of a compound shown to bind to and/or modulate the activity of heteromeric T1R2/T1R3 or a compound shown to bind to and/or modulate the activity of heteromeric T1R1/T1R3. In a preferred embodiment the counter-screen assay will fluorimetrically detect homomeric T1R2 activity by detecting intracellular calcium using a FLIPR system.
[0056] The terms "purified," "substantially purified," and "isolated" as used herein refer to the state of being free of other, dissimilar compounds with which the compound of the invention is normally associated in its natural state, so that the "purified," "substantially purified," and "isolated" subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample. In one preferred embodiment, these terms refer to the compound of the invention comprising at least 95% of the mass, by weight, of a given sample. As used herein, the terms "purified," "substantially purified," and "isolated," when referring to a nucleic acid or protein, of nucleic acids or proteins, also refers to a state of purification or concentration different than that which occurs naturally in the mammalian, especially human, body. Any degree of purification or concentration greater than that which occurs naturally in the mammalian, especially human, body, including (1) the purification from other associated structures or compounds or (2) the association with structures or compounds to which it is not normally associated in the mammalian, especially human, body, are within the meaning of "isolated." The nucleic acid or protein or classes of nucleic acids or proteins, described herein, may be isolated, or otherwise associated with structures or compounds to which they are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.
[0057] The term "nucleic acid" or "nucleic acid sequence" refers to a deoxy-ribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogs of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones (see e.g., Oligonucleotides and Analogues, a Practical Approach, ed. F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the N.Y. Academy of Sciences, Vol. 600, Eds. Baserga et al. (NYAS 1992); Milligan J. Med. Chem. 36:1923-1937 (1993); Antisense Research and Applications (1993, CRC Press), WO 97/03211; WO 96/39154; Mata, Toxicol. Appl. Pharmacol. 144:189-197 (1997); Strauss-Soukup, Biochemistry 36:8692-8698 (1997); Samstag, Antisense Nucleic Acid Drug Dev, 6:153-156 (1996)).
[0058] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating, e.g., sequences in which the third position of one or more selected codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
[0059] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
[0060] The term "plasma membrane translocation domain" or simply "translocation domain" means a polypeptide domain that, when incorporated into a polypeptide coding sequence, can with great efficiency "chaperone" or "translocate" the hybrid ("fusion") protein to the cell plasma membrane. For instance, a "translocation domain" may be derived from the amino terminus of the bovine rhodopsin receptor polypeptide, a 7-transmembrane receptor. However, rhodopsin from any mammal may be used, as can other translocation facilitating sequences. Thus, the translocation domain is particularly efficient in translocating 7-transmembrane fusion proteins to the plasma membrane, and a protein (e.g., a taste receptor polypeptide) comprising an amino terminal translocating domain may be transported to the plasma membrane more efficiently than without the domain.
[0061] The "translocation domain," "ligand-binding domain", and chimeric receptors compositions described herein also include "analogs," or "conservative variants" and "mimetics" ("peptidomimetics") with structures and activity that substantially correspond to the exemplary sequences. Thus, the terms "conservative variant" or "analog" or "mimetic" refer to a polypeptide which has a modified amino acid sequence, such that the change(s) do not substantially alter the polypeptide's (the conservative variant's) structure and/or activity, as defined herein. These include conservatively modified variations of an amino acid sequence, i.e., amino acid substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity.
[0062] More particularly, "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
[0063] For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
[0064] Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
[0065] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): ala/gly or ser; arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gln; ile/leu or val; leu/ile or val; lys/arg or gln or glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu. An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H. Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Vrlag (1979)). One of skill in the art will appreciate that the above-identified substitutions are not the only possible conservative substitutions. For example, for some purposes, one may regard all charged amino acids as conservative substitutions for each other whether they are positive or negative. In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered "conservatively modified variations."
[0066] The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound that has substantially the same structural and/or functional characteristics of the polypeptides, e.g., translocation domains, ligand-binding domains, or chimeric receptors of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogs of amino acids, or may be a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity.
[0067] As with polypeptides of the invention which are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, a helix conformation, and the like. A polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond ("peptide bond") linkages include, e.g., ketomethylene (e.g., --C.dbd.O--CH2- for --C.dbd.O)--NH--, aminomethylene (CH2-NH), ethylene, olefin (CH.dbd.CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY (1983)). A polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature.
[0068] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
[0069] A "labeled nucleic acid probe or oligonucleotide" is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
[0070] As used herein a "nucleic acid probe or oligonucleotide" is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, for example, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are optionally directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
[0071] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
[0072] A "promoter" is defined as an array of nucleic acid sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
[0073] As used herein, "recombinant" refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., "recombinant polynucleotide"), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide. "Recombinant means" also encompass the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of, e.g., inducible or constitutive expression of a fusion protein comprising a T1R protein according to the invention.
[0074] The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
[0075] The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10 degrees C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60 degrees C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5.times.SSC, and 1% SDS, incubating at 42 degrees C., or, 5.times.SSC, 1% SDS, incubating at 65 degrees C., with wash in 0.2.times.SSC, and 0.1% SDS at 65 degrees C. Such hybridizations and wash steps can be carried out for, e.g., 1, 2, 5, 10, 15, 30, 60; or more minutes.
[0076] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially related if the polypeptides which they encode are substantially related. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 degrees C., and a wash in 1.times.SSC at 45 degrees C. Such hybridizations and wash steps can be carried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
[0077] "Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
[0078] An "anti-T1R" antibody is an antibody or antibody fragment that specifically binds a polypeptide encoded by a T1R gene, cDNA, or a subsequence thereof.
[0079] The term "immunoassay" is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
[0080] The phrase "specifically (or selectively) binds" to an antibody or, "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to a T1R family member from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the T1R polypeptide or an immunogenic portion thereof and not with other proteins, except for orthologs or polymorphic variants and alleles of the T1R polypeptide. This selection may be achieved by subtracting out antibodies that cross-react with T1R molecules from other species or other T1R molecules. Antibodies can also be selected that recognize only T1R GPCR family members but not GPCRs from other families. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
[0081] The phrase "selectively associates with" refers to the ability of a nucleic acid to "selectively hybridize" with another as defined above, or the ability of an antibody to "selectively (or specifically) bind to a protein, as defined above.
[0082] The term "expression vector" refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome. The expression systems can have the ability to self-replicate or not, i.e., drive only transient expression in a cell. The term includes recombinant expression "cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid.
[0083] By "host cell" is meant a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa, U2OS, U2OS, NIH3T3 or MDCK cell, BHK cells, and the like, e.g., cultured cells, explants, and cells in vivo.
Detection of T1R Modulators
[0084] Exemplary compositions and methods for determining whether a test compound specifically binds to a T1R2/T1R3 or T1R2/T1R3 heteromer or a homomeric T1R, e.g., T1R1, T1R2 or T1R3, are described below. Many aspects of cell physiology can be monitored to assess the effect of ligand binding to a T1R polypeptide of the invention. These assays may be performed on intact cells expressing at least one T1R receptor, on permeabilized cells, or on membrane fractions produced by standard methods.
[0085] Taste receptors bind tastants and initiate the transduction of chemical stimuli into electrical signals. An activated or inhibited G protein will in turn alter the properties of target enzymes, channels, and other effector proteins. Some examples are the activation of cGMP phosphodiesterase by transducin in the visual system, adenylate cyclase by the stimulatory G protein, phospholipase C by Gq and other cognate G proteins, and modulation of diverse channels by Gi and other G proteins. Downstream consequences can also be examined such as generation of diacyl glycerol and IP3 by phospholipase C, and in turn, for calcium mobilization by IP3.
[0086] The T1R proteins or heteromers used in these assays will typically comprise a human or rodent T1R or heteromer, most typically human T1R2/T1R3 or T1R1/T1R3 or a homomeric T1R1 or T1R2 as defined herein. As noted, this includes T1R polypeptide fragments, chimeras, and conservatively modified variants thereof. These T1R polypeptides may be expressed in recombinant or endogenous cells, e.g., isolated human cells.
[0087] Alternatively, the T1R proteins or polypeptides of the assay can be derived from a eukaryote host cell and can include an amino acid subsequence having amino acid sequence identity to a T1R polypeptide contained in the Sequence Listing or known in the art or fragments or conservatively modified variants thereof. Generally, the amino acid sequence identity will be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Optionally, the T1R proteins or polypeptides of the assays can comprise a domain of a T1R protein, such as an extracellular domain, transmembrane region, transmembrane domain, cytoplasmic domain, ligand-binding domain, and the like. Further, as described above, the T1R protein or a domain thereof can be covalently linked to a heterologous protein to create a chimeric protein used in the assays described herein.
[0088] Modulators of T1R receptor activity are tested using T1R proteins or polypeptides as described above, either recombinant or naturally occurring. The T1R proteins or polypeptides can be isolated, co-expressed in a cell, co-expressed in a membrane derived from a cell, co-expressed in tissue or in an animal, either recombinant or naturally occurring. For example, tongue slices, dissociated cells from a tongue, transformed cells, or membranes can be used. Modulation can be tested using one of the in vitro or in vivo assays described herein.
[0089] In Vitro Binding Assays
[0090] Taste transduction can also be examined in vitro with soluble or solid state reactions, using hetero-oligomeric complexes of the T1R polypeptides of the invention. In a particular embodiment, hetero-oligomeric complexes of T1R ligand-binding domains can be used in vitro in soluble or solid state reactions to assay for ligand binding.
[0091] Ligand binding to a T1R2/T1R3 or T1R1/T1R3 heteromer or a homomeric T1R polypeptide can be tested in solution, in a bilayer membrane, optionally attached to a solid phase, in a lipid monolayer, or in vesicles. Binding of a modulator can be tested using, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index) hydrodynamic (e.g., shape), chromatographic, or solubility properties. Preferred binding assays of the invention are biochemical binding assays that use recombinant soluble T1R polypeptides.
[0092] Receptor-G protein interactions can also be examined. For example, binding of the G protein to the receptor complex, or its release from the receptor complex can be examined. More particularly, in the absence of GTP, an activator will lead to the formation of a tight complex of a G protein (all three subunits) with the receptor. This complex can be detected in a variety of ways, as noted above. Such an assay can be modified to search for inhibitors, e.g., by adding an activator to the receptor and G protein in the absence of GTP, which form a tight complex, and then screen for inhibitors by looking at dissociation of the receptor-G protein complex. In the presence of GTP, release of the a subunit of the G protein from the other two G protein subunits serves as a criterion of activation. An activated or inhibited G protein will in turn alter the properties of target enzymes, channels, and other effector proteins.
[0093] In another embodiment of the invention, a GTP .gamma..sup.35S assay may be used. As described above, upon activation of a GPCR, the G a subunit of the G protein complex is stimulated to exchange bound GDP for GTP. Ligand-mediated stimulation of G protein exchange activity can be measured in a biochemical assay measuring the binding of added radioactively labeled GTP .gamma..sup.35S to the G protein in the presence of a putative ligand. Typically, membranes containing the chemosensory receptor of interest are mixed with a complex of G proteins. Potential inhibitors and/or activators and GTP.gamma. 35S are added to the assay, and binding of GTP .gamma..sup.35S to the G protein is measured. Binding can be measured by liquid scintillation counting or by any other means known in the art, including scintillation proximity assays (SPA). In other assays formats, fluorescently labeled GTP.gamma.35S can be utilized.
Fluorescence Polarization Assays
[0094] In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels. This section describes how fluorescence polarization can be used in a simple and quantitative way to measure the binding of ligands to the T1R polypeptides of the invention.
[0095] When a fluorescently labeled molecule is excited with plane polarized light, it emits light that has a degree of polarization that is inversely proportional to its molecular rotation. Large fluorescently labeled molecules remain relatively stationary during the excited state (4 nanoseconds in the case of fluorescein) and the polarization of the light remains relatively constant between excitation and emission. Small fluorescently labeled molecules rotate rapidly during the excited state and the polarization changes significantly between excitation and emission. Therefore, small molecules have low polarization values and large molecules have high polarization values. For example, a single-stranded fluorescein-labeled oligonucleotide has a relatively low polarization value but when it is hybridized to a complementary strand, it has a higher polarization value. When using FP to detect and monitor tastant-binding which may activate or inhibit the chemosensory receptors of the invention, fluorescence-labeled tastants or auto-fluorescent tastants may be used. For example, fluorescence polarization has been used to measure enzymatic cleavage of large fluorescein labeled polymers by proteases, DNases, and RNases. It also has been used to measure equilibrium binding for protein/protein interactions, antibody/antigen binding, and protein/DNA binding.
Solid State and Soluble High Throughput Assays
[0096] In yet another embodiment, the invention provides soluble assays using T1R heteromers or homomeric T1R polypeptides; or a cell or tissue co-expressing T1R polypeptides or only one T1R polypeptide. In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the T1R polypeptides, or cell or tissue expressing the T1R polypeptides is attached to a solid phase substrate.
[0097] In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 1000 to about 1500 different compounds. It is also possible to assay multiple compounds in each plate well. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention. Also, microfluidic reagent manipulation may be used.
[0098] The molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non-covalent linkage, e.g., via a tag. The tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest (e.g., the taste transduction molecule of interest) is attached to the solid support by interaction of the tag and the tag binder.
[0099] A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.). Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).
[0100] Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs. For example, agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherein family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.
[0101] Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
[0102] Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to persons of skill in the art. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
[0103] Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth., 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank & Doring, Tetrahedron, 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry, 39(4):718-719 (1993); and Kozal et al., Nature Medicine, 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
Cell-Based Binding Assays
[0104] T1R proteins or polypeptides are expressed in a eukaryotic or non-eukaryotic cell, preferably a mammalian cell or an oocyte. The subject T1R polypeptides can be expressed in any eukaryotic cell, such as U2OS, U2OS, NIH3T3 or MDCK cells or other mammalian cells. Preferably, the cells comprise a functional G protein, e.g., G.alpha.15, or a chimeric G protein such as a chimera of G16 or G15 and gustducin or transducin that is capable of coupling the chimeric receptor to an intracellular signaling pathway or to a signaling protein such as phospholipase C. Activation of such chimeric receptors in such cells can be detected using any standard method, such as by detecting changes in intracellular calcium by detecting FURA-2 dependent fluorescence in the cell.
[0105] Activated GPCR receptors become substrates for kinases that phosphorylate the C-terminal tail of the receptor (and possibly other sites as well). Thus, activators will promote the transfer of 32P from .gamma.-labeled GTP to the receptor, which can be assayed with a scintillation counter. The phosphorylation of the C-terminal tail will promote the binding of arrestin-like proteins and will interfere with the binding of G proteins. The kinase/arrestin pathway plays a key role in the desensitization of many GPCR receptors. For example, compounds that modulate the duration a taste receptor stays active would be useful as a means of prolonging a desired taste or cutting off an unpleasant one. For a general review of GPCR signal transduction and methods of assaying signal transduction, see, e.g., Methods in Enzymology, vols. 237 and 238 (1994) and volume 96 (1983); Bourne et al., Nature, 10:349:117-27 (1991); Bourne et al., Nature, 348:125-32 (1990); Pitcher et al., Annu. Rev. Biochem., 67:653-92 (1998).
[0106] T1R modulation may be assayed by comparing the response of T1R polypeptides treated with a putative T1R modulator to the response of an untreated control sample. Such putative T1R modulators can include tastants that either inhibit or activate T1R polypeptide activity. In one embodiment, control samples (untreated with activators or inhibitors) are assigned a relative T1R activity value of 100. Inhibition of a T1R polypeptide is achieved when the T1R activity value relative to the control is about 90%, optionally 50%, optionally 25-0%. Activation of a T1R polypeptide is achieved when the T1R activity value relative to the control is 110%, optionally 150%, 200-500%, or 1000-2000%.
[0107] Changes in ion flux may be assessed by determining changes in ionic polarization (i.e., electrical potential) of the cell or membrane expressing a T1R polypeptide. One means to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques (see, e.g., the "cell-attached" mode, the "inside-out" mode, and the "whole cell" mode, e.g., Ackerman et al., New Engl. J. Med., 336:1575-1595 (1997)). Whole cell currents are conveniently determined using the standard. Other known assays include: radiolabeled ion flux assays and fluorescence assays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al., J. Membrane Biol., 88:67-75 (1988); Gonzales & Tsien, Chem. Biol., 4:269277 (1997); Daniel et al., J. Pharmacol. Meth., 25:185-193 (1991); Holevinsky et al., J. Membrane Biology, 137:59-70 (1994)). Generally, the compounds to be tested are present in the range from 1 pM to 100 mM.
[0108] The effects of the test compounds upon the function of the polypeptides can be measured by examining any of the parameters described above. Any suitable physiological change that affects GPCR activity can be used to assess the influence of a test compound on the polypeptides of this invention. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as transmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca.sup.2+, IP3, cGMP, or cAMP.
[0109] Other assays for GPCRs include cells that are loaded with ion or voltage sensitive dyes to report receptor activity. Assays for determining activity of such receptors can also use known agonists and antagonists for other G protein-coupled receptors as negative or positive controls to assess activity of tested compounds. In assays for identifying modulatory compounds (e.g. agonists, antagonists), changes in the level of ions in the cytoplasm or membrane voltage will be monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively. Among the ion-sensitive indicators and voltage probes that may be employed are those disclosed in the Molecular Probes 1997 Catalog. For G protein-coupled receptors, promiscuous G proteins such as G.alpha.15 and G.alpha.16 or the afore-mentioned chimeric G proteins can be used in the assay of choice (Wilkie et al., Proc. Nat'l Acad. Sci., 88:10049-10053 (1991)). Such promiscuous G proteins allow coupling of a wide range of receptors.
[0110] Receptor activation typically initiates subsequent intracellular events, e.g., increases in second messengers such as IP3, which releases intracellular stores of calcium ions. Activation of some G protein-coupled receptors stimulates the formation of inositol triphosphate (IP3) through phospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge & Irvine, Nature, 312:315-21 (1984)). IP3 in turn stimulates the release of intracellular calcium ion stores. Thus, a change in cytoplasmic calcium ion levels, or a change in second messenger levels such as IP3 can be used to assess G protein-coupled receptor function. Cells expressing such G protein-coupled receptors may exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it may be desirable although not necessary to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such as EGTA, to distinguish fluorescence response resulting from calcium release from internal stores.
[0111] Other assays can involve determining the activity of receptors which, when activated, result in a change in the level of intracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymes such as adenylate cyclase. There are cyclic nucleotide-gated ion channels, e.g., rod photoreceptor cell channels and olfactory neuron channels that are permeable to cations upon activation by binding of cAMP or cGMP (see, e.g., Altenhofen et al., Proc. Nat'l Acad. Sci., 88:9868-9872 (1991) and Dhallan et al., Nature, 347:184-187 (1990)). In cases where activation of the receptor results in a decrease in cyclic nucleotide levels, it may be preferable to expose the cells to agents that increase intracellular cyclic nucleotide levels, e.g., forskolin, prior to adding a receptor-activating compound to the cells in the assay. Cells for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-crated ion channel, GPCR phosphatase and DNA encoding a receptor (e.g., certain glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like), which, when activated, causes a change in cyclic nucleotide levels in the cytoplasm.
[0112] In a preferred embodiment, T1R polypeptide activity is measured by co-expressing T1R2 and T1R3 nucleic acids or T1R1 and T1R3 nucleic acids in a heterologous cell with a promiscuous or chimeric G protein that links the receptor to a phospholipase C signal transduction pathway (see Offermanns & Simon, J. Biol. Chem., 270:15175-15180 (1995)). Optionally the cell line is U2OS, U2OS, NIH3T3 or MDCK cell line or other mammalian cell that does not naturally express T1R genes and the G protein is G.alpha.15 or G.alpha.16 (Offermanns & Simon, supra), gustducin, transducin, or is a chimeric G protein containing any of the foregoing. Modulation of taste transduction is assayed by measuring changes in intracellular Ca.sup.2+ levels, which change in response to modulation of the T1R signal transduction pathway via administration of a molecule that associates with T1R polypeptides. Changes in Ca.sup.2+ levels are optionally measured using fluorescent Ca.sup.2+ indicator dyes and fluorimetric imaging. Preferably this is effected using a FLIPR detection system.
[0113] In one embodiment, the changes in intracellular cAMP or cGMP can be measured using immunoassays. The method described in Offemmanns & Simon, J. Bio. Chem., 270:15175-15180 (1995), may be used to determine the level of cAMP. Also, the method described in Felley-Bosco et al., Am. J. Resp. Cell and Mol. Biol., 11:159-164 (1994), may be used to determine the level of cGMP. Further, an assay kit for measuring cAMP and/or cGMP is described in U.S. Pat. No. 4,115,538, herein incorporated by reference.
[0114] In another embodiment, phosphatidyl inositol (PI) hydrolysis can be analyzed according to U.S. Pat. No. 5,436,128, herein incorporated by reference. Briefly, the assay involves labeling of cells with 3H-myoinositol for 48 or more hrs. The labeled cells are treated with a test compound for one hour. The treated cells are lysed and extracted in chloroform-methanol-water after which the inositol phosphates were separated by ion exchange chromatography and quantified by scintillation counting. Fold stimulation is determined by calculating the ratio of cpm in the presence of agonist, to cpm in the presence of buffer control. Likewise, fold inhibition is determined by calculating the ratio of cpm in the presence of antagonist, to cpm in the presence of buffer control (which may or may not contain an agonist).
[0115] In another embodiment, transcription levels can be measured to assess the effects of a test compound on signal transduction. A host cell containing T1R polypeptides of interest is contacted with a test compound for a sufficient time to effect any interactions, and then the level of gene expression is measured. The amount of time to effect such interactions may be empirically determined, such as by running a time course and measuring the level of transcription as a function of time. The amount of transcription may be measured by using any method known to those of skill in the art to be suitable. For example, mRNA expression of the protein of interest may be detected using northern blots or their polypeptide products may be identified using immunoassays. Alternatively, transcription based assays using reporter gene may be used as described in U.S. Pat. No. 5,436,128, herein incorporated by reference. The reporter genes can be, e.g., chloramphenicol acetyltransferase, luciferase, 3'-galactosidase and alkaline phosphatase. Furthermore, the protein of interest can be used as an indirect reporter via attachment to a second reporter such as green fluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology, 15:961-964 (1997)).
[0116] The amount of transcription is then compared to the amount of transcription in either the same cell in the absence of the test compound, or it may be compared with the amount of transcription in a substantially identical cell that lacks the T1R polypeptide of interest. A substantially identical cell may be derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. Any difference in the amount of transcription indicates that the test compound has in some manner altered the activity of the T1R polypeptides of interest.
[0117] The invention is further described by the following examples.
EXAMPLES
[0118] In all of the examples below, assays were conducted using mammalian cells stably expressing hT1R2 and hT1R3, as well as mammalian cells transiently expressing hT1R2 alone or a control Mock vector using the FLIPR system to evaluate T1R2/T1R3 or homomeric T1R2 receptor activation. All of the cells express the chimeric G protein G16gust25. The production of cells stably expressing hT1R2 and hT1R3, and G16gust25 has been disclosed in earlier Senomyx patents. The hT1R2 gene was transduced into cells expressing G16gust25 and 48 hours later, test compounds or sweeteners were evaluated the same way as assays conducted using cells expressing hT1R2 and hT1R3, as well as Mock transfected cells using the FLIPR system.
Example 1
[0119] Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound A, a sweet taste receptor agonist known to bind to the transmembrane domain of hT1R2. Compound A was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay producing kinetics of calcium mobilization that are typical of GPCR activation. These results are contained in FIG. 1.
Example 2
[0120] Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound B, a sweet taste receptor agonist. Compound B was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay. These results are contained in FIG. 2.
Example 3
[0121] Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound C, a sweet taste receptor agonist. Compound C was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay. These results are contained in FIG. 3.
Example 4
[0122] Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound D, a sweet taste receptor agonist. Compound D was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay. These results are contained in FIG. 4.
Example 5
[0123] Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by Perillartine, a sweet taste receptor agonist. Perillartine was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay. These results are contained in FIG. 5.
Example 6
[0124] Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by P-4000, a sweet taste receptor agonist. P-4000 was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay. These results are contained in FIG. 6.
Example 7
[0125] Seventeen compounds or sweeteners identified as agonists in the hT1R2/hT1R3 assay and showing a good correlation between taste data and assay data (within a 95% confidence interval) were inactive in the hT1R2 assay. Conversely, six compounds identified as agonists in the hT1R2/hT1R3 assay but ultimately found to behave poorly in human taste tests or being tasteless were active in the hT1R2 assay.
[0126] FIG. 7 summarizes the potency of these 17 compounds at different molar ratios relative to sucrose in the hT1R2/hT1R3 assay. Based on these results it can be seen that assays conducted using cells that express T1R2 alone (i.e., in the absence of T1R3), e.g., FLIPR based assays with compounds identified as putative sweet taste modulators based on their modulation of the activity of T1R2/T1R3 is an effective means for potentially minimizing or even eliminating false positives. Given the time and expense associated with identifying and producing optimized compounds suitable for use in products for human consumption this is a significant improvement that should lead to the development of novel sweet taste modulators.
[0127] The exemplified counter-screen assay in addition may be further optimized to use as a counter-screen, on a regular basis, to eliminate false positives, such as by producing and using cells that stably express T1R2 alone (i.e., in the absence of T1R3) and a suitable G protein such as G16gust25, G15gust25, G16trans44 or G16trans44.
Sequence CWU
1
1
311858PRTHomo sapiens 1Met Pro Gly Leu Ala Ile Leu Gly Leu Ser Leu Ala Ala
Phe Leu Glu1 5 10 15Leu
Gly Met Gly Ser Ser Leu Cys Leu Ser Gln Gln Phe Lys Ala Gln 20
25 30Gly Asp Tyr Ile Leu Gly Gly Leu
Phe Pro Leu Gly Thr Thr Glu Glu 35 40
45Ala Thr Leu Asn Gln Arg Thr Gln Pro Asn Gly Ile Leu Cys Thr Arg
50 55 60Phe Ser Pro Leu Gly Leu Phe Leu
Ala Met Ala Met Lys Met Ala Val65 70 75
80Glu Glu Ile Asn Asn Gly Ser Ala Leu Leu Pro Gly Leu
Arg Leu Gly 85 90 95Tyr
Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Thr Met Lys Pro
100 105 110Ser Leu Met Phe Met Ala Lys
Val Gly Ser Gln Ser Ile Ala Ala Tyr 115 120
125Cys Asn Tyr Thr Gln Tyr Gln Pro Arg Val Leu Ala Val Ile Gly
Pro 130 135 140His Ser Ser Glu Leu Ala
Leu Ile Thr Gly Lys Phe Phe Ser Phe Phe145 150
155 160Leu Met Pro Gln Val Ser Tyr Ser Ala Ser Met
Asp Arg Leu Ser Asp 165 170
175Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val
180 185 190Gln Leu Gln Ala Val Val
Thr Leu Leu Gln Asn Phe Ser Trp Asn Trp 195 200
205Val Ala Ala Leu Gly Ser Asp Asp Asp Tyr Gly Arg Glu Gly
Leu Ser 210 215 220Ile Phe Ser Gly Leu
Ala Asn Ser Arg Gly Ile Cys Ile Ala His Glu225 230
235 240Gly Leu Val Pro Gln His Asp Thr Ser Gly
Gln Gln Leu Gly Lys Val 245 250
255Val Asp Val Leu Arg Gln Val Asn Gln Ser Lys Val Gln Val Val Val
260 265 270Leu Phe Ala Ser Ala
Arg Ala Val Tyr Ser Leu Phe Ser Tyr Ser Ile 275
280 285Leu His Asp Leu Ser Pro Lys Val Trp Val Ala Ser
Glu Ser Trp Leu 290 295 300Thr Ser Asp
Leu Val Met Thr Leu Pro Asn Ile Ala Arg Val Gly Thr305
310 315 320Val Leu Gly Phe Leu Gln Arg
Gly Ala Leu Leu Pro Glu Phe Ser His 325
330 335Tyr Val Glu Thr Arg Leu Ala Leu Ala Ala Asp Pro
Thr Phe Cys Ala 340 345 350Ser
Leu Lys Ala Glu Leu Asp Leu Glu Glu Arg Val Met Gly Pro Arg 355
360 365Cys Ser Gln Cys Asp Tyr Ile Met Leu
Gln Asn Leu Ser Ser Gly Leu 370 375
380Met Gln Asn Leu Ser Ala Gly Gln Leu His His Gln Ile Phe Ala Thr385
390 395 400Tyr Ala Ala Val
Tyr Ser Val Ala Gln Ala Leu His Asn Thr Leu Gln 405
410 415Cys Asn Val Ser His Cys His Thr Ser Glu
Pro Val Gln Pro Trp Gln 420 425
430Leu Leu Glu Asn Met Tyr Asn Met Ser Phe Arg Ala Arg Asp Leu Thr
435 440 445Leu Gln Phe Asp Ala Lys Gly
Ser Val Asp Met Glu Tyr Asp Leu Lys 450 455
460Met Trp Val Trp Gln Ser Pro Thr Pro Val Leu His Thr Val Gly
Thr465 470 475 480Phe Asn
Gly Thr Leu Gln Leu Gln His Ser Lys Met Tyr Trp Pro Gly
485 490 495Asn Gln Val Pro Val Ser Gln
Cys Ser Arg Gln Cys Lys Asp Gly Gln 500 505
510Val Arg Arg Val Lys Gly Phe His Ser Cys Cys Tyr Asp Cys
Val Asp 515 520 525Cys Lys Ala Gly
Ser Tyr Arg Lys His Pro Asp Asp Phe Thr Cys Thr 530
535 540Pro Cys Gly Lys Asp Gln Trp Ser Pro Glu Lys Ser
Thr Thr Cys Leu545 550 555
560Pro Arg Arg Pro Lys Phe Leu Ala Trp Gly Glu Pro Ala Val Leu Ser
565 570 575Leu Leu Leu Leu Leu
Cys Leu Val Leu Gly Leu Thr Leu Ala Ala Leu 580
585 590Gly Leu Phe Val His Tyr Trp Asp Ser Pro Leu Val
Gln Ala Ser Gly 595 600 605Gly Ser
Leu Phe Cys Phe Gly Leu Ile Cys Leu Gly Leu Phe Cys Leu 610
615 620Ser Val Leu Leu Phe Pro Gly Arg Pro Arg Ser
Ala Ser Cys Leu Ala625 630 635
640Gln Gln Pro Met Ala His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu
645 650 655Phe Leu Gln Ala
Ala Glu Ile Phe Val Glu Ser Glu Leu Pro Leu Ser 660
665 670Trp Ala Asn Trp Leu Cys Ser Tyr Leu Arg Gly
Pro Trp Ala Trp Leu 675 680 685Val
Val Leu Leu Ala Thr Leu Val Glu Ala Ala Leu Cys Ala Trp Tyr 690
695 700Leu Met Ala Phe Pro Pro Glu Val Val Thr
Asp Trp Gln Val Leu Pro705 710 715
720Thr Glu Val Leu Glu His Cys Arg Met Arg Ser Trp Val Ser Leu
Gly 725 730 735Leu Val His
Ile Thr Asn Ala Val Leu Ala Phe Leu Cys Phe Leu Gly 740
745 750Thr Phe Leu Val Gln Ser Gln Pro Gly Arg
Tyr Asn Arg Ala Arg Gly 755 760
765Leu Thr Phe Ala Met Leu Ala Tyr Phe Ile Ile Trp Val Ser Phe Val 770
775 780Pro Leu Leu Ala Asn Val Gln Val
Ala Tyr Gln Pro Ala Val Gln Met785 790
795 800Gly Ala Ile Leu Phe Cys Ala Leu Gly Ile Leu Ala
Thr Phe His Leu 805 810
815Pro Lys Cys Tyr Val Leu Leu Trp Leu Pro Glu Leu Asn Thr Gln Glu
820 825 830Phe Phe Leu Gly Arg Ser
Pro Lys Glu Ala Ser Asp Gly Asn Ser Gly 835 840
845Ser Ser Glu Ala Thr Arg Gly His Ser Glu 850
8552841PRTHomo sapiens 2Met Leu Leu Cys Thr Ala Arg Leu Val Gly Leu
Gln Leu Leu Ile Ser1 5 10
15Cys Cys Trp Ala Phe Ala Cys His Ser Thr Glu Ser Ser Pro Asp Phe
20 25 30Thr Leu Pro Gly Asp Tyr Leu
Leu Ala Gly Leu Phe Pro Leu His Ser 35 40
45Gly Cys Leu Gln Val Arg His Arg Pro Glu Val Thr Leu Cys Asp
Arg 50 55 60Ser Cys Ser Phe Asn Glu
His Gly Tyr His Leu Phe Gln Ala Met Arg65 70
75 80Leu Gly Val Glu Glu Ile Asn Asn Ser Thr Ala
Leu Leu Pro Asn Ile 85 90
95Thr Leu Gly Tyr Gln Leu Tyr Asp Val Cys Ser Asp Ser Ala Asn Val
100 105 110Tyr Ala Thr Leu Arg Val
Leu Ser Leu Pro Gly Gln His His Ile Glu 115 120
125Leu Gln Gly Asp Leu Leu His Tyr Ser Pro Thr Val Leu Ala
Val Ile 130 135 140Gly Pro Asp Ser Thr
Asn Arg Ala Ala Thr Thr Ala Ala Leu Leu Ser145 150
155 160Pro Phe Leu Val Pro Met Ile Ser Tyr Ala
Ala Ser Ser Glu Thr Leu 165 170
175Ser Val Lys Arg Gln Tyr Pro Ser Phe Leu Arg Thr Ile Pro Asn Asp
180 185 190Lys Tyr Gln Val Glu
Thr Met Val Leu Leu Leu Gln Lys Phe Gly Trp 195
200 205Thr Trp Ile Ser Leu Val Gly Ser Ser Asp Asp Tyr
Gly Gln Leu Gly 210 215 220Val Gln Ala
Leu Glu Asn Gln Ala Thr Gly Gln Gly Ile Cys Ile Ala225
230 235 240Phe Lys Asp Ile Met Pro Phe
Ser Ala Gln Val Gly Asp Glu Arg Met 245
250 255Gln Cys Leu Met Arg His Leu Ala Gln Ala Gly Ala
Thr Val Val Val 260 265 270Val
Phe Ser Ser Arg Gln Leu Ala Arg Val Phe Phe Glu Ser Val Val 275
280 285Leu Thr Asn Leu Thr Gly Lys Val Trp
Val Ala Ser Glu Ala Trp Ala 290 295
300Leu Ser Arg His Ile Thr Gly Val Pro Gly Ile Gln Arg Ile Gly Met305
310 315 320Val Leu Gly Val
Ala Ile Gln Lys Arg Ala Val Pro Gly Leu Lys Ala 325
330 335Phe Glu Glu Ala Tyr Ala Arg Ala Asp Lys
Lys Ala Pro Arg Pro Cys 340 345
350His Lys Gly Ser Trp Cys Ser Ser Asn Gln Leu Cys Arg Glu Cys Gln
355 360 365Ala Phe Met Ala His Thr Met
Pro Lys Leu Lys Ala Phe Ser Met Ser 370 375
380Ser Ala Tyr Asn Ala Tyr Arg Ala Val Tyr Ala Val Ala His Gly
Leu385 390 395 400His Gln
Leu Leu Gly Cys Ala Ser Gly Ala Cys Ser Arg Gly Arg Val
405 410 415Tyr Pro Trp Gln Leu Leu Glu
Gln Ile His Lys Val His Phe Leu Leu 420 425
430His Lys Asp Thr Val Ala Phe Asn Asp Asn Arg Asp Pro Leu
Ser Ser 435 440 445Tyr Asn Ile Ile
Ala Trp Asp Trp Asn Gly Pro Lys Trp Thr Phe Thr 450
455 460Val Leu Gly Ser Ser Thr Trp Ser Pro Val Gln Leu
Asn Ile Asn Glu465 470 475
480Thr Lys Ile Gln Trp His Gly Lys Asp Asn Gln Val Pro Lys Ser Val
485 490 495Cys Ser Ser Asp Cys
Leu Glu Gly His Gln Arg Val Val Thr Gly Phe 500
505 510His His Cys Cys Phe Glu Cys Val Pro Cys Gly Ala
Gly Thr Phe Leu 515 520 525Asn Lys
Ser Asp Leu Tyr Arg Cys Gln Pro Cys Gly Lys Glu Glu Trp 530
535 540Ala Pro Glu Gly Ser Gln Thr Cys Phe Pro Arg
Thr Val Val Phe Leu545 550 555
560Ala Leu Arg Glu His Thr Ser Trp Val Leu Leu Ala Ala Asn Thr Leu
565 570 575Leu Leu Leu Leu
Leu Leu Gly Thr Ala Gly Leu Phe Ala Trp His Leu 580
585 590Asp Thr Pro Val Val Arg Ser Ala Gly Gly Arg
Leu Cys Phe Leu Met 595 600 605Leu
Gly Ser Leu Ala Ala Gly Ser Gly Ser Leu Tyr Gly Phe Phe Gly 610
615 620Glu Pro Thr Arg Pro Ala Cys Leu Leu Arg
Gln Ala Leu Phe Ala Leu625 630 635
640Gly Phe Thr Ile Phe Leu Ser Cys Leu Thr Val Arg Ser Phe Gln
Leu 645 650 655Ile Ile Ile
Phe Lys Phe Ser Thr Lys Val Pro Thr Phe Tyr His Ala 660
665 670Trp Val Gln Asn His Gly Ala Gly Leu Phe
Val Met Ile Ser Ser Ala 675 680
685Ala Gln Leu Leu Ile Cys Leu Thr Trp Leu Val Val Trp Thr Pro Leu 690
695 700Pro Ala Arg Glu Tyr Gln Arg Phe
Pro His Leu Val Met Leu Glu Cys705 710
715 720Thr Glu Thr Asn Ser Leu Gly Phe Ile Leu Ala Phe
Leu Tyr Asn Gly 725 730
735Leu Leu Ser Ile Ser Ala Phe Ala Cys Ser Tyr Leu Gly Lys Asp Leu
740 745 750Pro Glu Asn Tyr Asn Glu
Ala Lys Cys Val Thr Phe Ser Leu Leu Phe 755 760
765Asn Phe Val Ser Trp Ile Ala Phe Phe Thr Thr Ala Ser Val
Tyr Asp 770 775 780Gly Lys Tyr Leu Pro
Ala Ala Asn Met Met Ala Gly Leu Ser Ser Leu785 790
795 800Ser Ser Gly Phe Gly Gly Tyr Phe Leu Pro
Lys Cys Tyr Val Ile Leu 805 810
815Cys Arg Pro Asp Leu Asn Ser Thr Glu His Phe Gln Ala Ser Ile Gln
820 825 830Asp Tyr Thr Arg Arg
Cys Gly Ser Thr 835 8403839PRTHomo sapiens 3Met
Gly Pro Arg Ala Lys Thr Ile Cys Ser Leu Phe Phe Leu Leu Trp1
5 10 15Val Leu Ala Glu Pro Ala Glu
Asn Ser Asp Phe Tyr Leu Pro Gly Asp 20 25
30Tyr Leu Leu Gly Gly Leu Phe Ser Leu His Ala Asn Met Lys
Gly Ile 35 40 45Val His Leu Asn
Phe Leu Gln Val Pro Met Cys Lys Glu Tyr Glu Val 50 55
60Lys Val Ile Gly Tyr Asn Leu Met Gln Ala Met Arg Phe
Ala Val Glu65 70 75
80Glu Ile Asn Asn Asp Ser Ser Leu Leu Pro Gly Val Leu Leu Gly Tyr
85 90 95Glu Ile Val Asp Val Cys
Tyr Ile Ser Asn Asn Val Gln Pro Val Leu 100
105 110Tyr Phe Leu Ala His Glu Asp Asn Leu Leu Pro Ile
Gln Glu Asp Tyr 115 120 125Ser Asn
Tyr Ile Ser Arg Val Val Ala Val Ile Gly Pro Asp Asn Ser 130
135 140Glu Ser Val Met Thr Val Ala Asn Phe Leu Ser
Leu Phe Leu Leu Pro145 150 155
160Gln Ile Thr Tyr Ser Ala Ile Ser Asp Glu Leu Arg Asp Lys Val Arg
165 170 175Phe Pro Ala Leu
Leu Arg Thr Thr Pro Ser Ala Asp His His Val Glu 180
185 190Ala Met Val Gln Leu Met Leu His Phe Arg Trp
Asn Trp Ile Ile Val 195 200 205Leu
Val Ser Ser Asp Thr Tyr Gly Arg Asp Asn Gly Gln Leu Leu Gly 210
215 220Glu Arg Val Ala Arg Arg Asp Ile Cys Ile
Ala Phe Gln Glu Thr Leu225 230 235
240Pro Thr Leu Gln Pro Asn Gln Asn Met Thr Ser Glu Glu Arg Gln
Arg 245 250 255Leu Val Thr
Ile Val Asp Lys Leu Gln Gln Ser Thr Ala Arg Val Val 260
265 270Val Val Phe Ser Pro Asp Leu Thr Leu Tyr
His Phe Phe Asn Glu Val 275 280
285Leu Arg Gln Asn Phe Thr Gly Ala Val Trp Ile Ala Ser Glu Ser Trp 290
295 300Ala Ile Asp Pro Val Leu His Asn
Leu Thr Glu Leu Gly His Leu Gly305 310
315 320Thr Phe Leu Gly Ile Thr Ile Gln Ser Val Pro Ile
Pro Gly Phe Ser 325 330
335Glu Phe Arg Glu Trp Gly Pro Gln Ala Gly Pro Pro Pro Leu Ser Arg
340 345 350Thr Ser Gln Ser Tyr Thr
Cys Asn Gln Glu Cys Asp Asn Cys Leu Asn 355 360
365Ala Thr Leu Ser Phe Asn Thr Ile Leu Arg Leu Ser Gly Glu
Arg Val 370 375 380Val Tyr Ser Val Tyr
Ser Ala Val Tyr Ala Val Ala His Ala Leu His385 390
395 400Ser Leu Leu Gly Cys Asp Lys Ser Thr Cys
Thr Lys Arg Val Val Tyr 405 410
415Pro Trp Gln Leu Leu Glu Glu Ile Trp Lys Val Asn Phe Thr Leu Leu
420 425 430Asp His Gln Ile Phe
Phe Asp Pro Gln Gly Asp Val Ala Leu His Leu 435
440 445Glu Ile Val Gln Trp Gln Trp Asp Arg Ser Gln Asn
Pro Phe Gln Ser 450 455 460Val Ala Ser
Tyr Tyr Pro Leu Gln Arg Gln Leu Lys Asn Ile Gln Asp465
470 475 480Ile Ser Trp His Thr Val Asn
Asn Thr Ile Pro Met Ser Met Cys Ser 485
490 495Lys Arg Cys Gln Ser Gly Gln Lys Lys Lys Pro Val
Gly Ile His Val 500 505 510Cys
Cys Phe Glu Cys Ile Asp Cys Leu Pro Gly Thr Phe Leu Asn His 515
520 525Thr Glu Asp Glu Tyr Glu Cys Gln Ala
Cys Pro Asn Asn Glu Trp Ser 530 535
540Tyr Gln Ser Glu Thr Ser Cys Phe Lys Arg Gln Leu Val Phe Leu Glu545
550 555 560Trp His Glu Ala
Pro Thr Ile Ala Val Ala Leu Leu Ala Ala Leu Gly 565
570 575Phe Leu Ser Thr Leu Ala Ile Leu Val Ile
Phe Trp Arg His Phe Gln 580 585
590Thr Pro Ile Val Arg Ser Ala Gly Gly Pro Met Cys Phe Leu Met Leu
595 600 605Thr Leu Leu Leu Val Ala Tyr
Met Val Val Pro Val Tyr Val Gly Pro 610 615
620Pro Lys Val Ser Thr Cys Leu Cys Arg Gln Ala Leu Phe Pro Leu
Cys625 630 635 640Phe Thr
Ile Cys Ile Ser Cys Ile Ala Val Arg Ser Phe Gln Ile Val
645 650 655Cys Ala Phe Lys Met Ala Ser
Arg Phe Pro Arg Ala Tyr Ser Tyr Trp 660 665
670Val Arg Tyr Gln Gly Pro Tyr Val Ser Met Ala Phe Ile Thr
Val Leu 675 680 685Lys Met Val Ile
Val Val Ile Gly Met Leu Ala Thr Gly Leu Ser Pro 690
695 700Thr Thr Arg Thr Asp Pro Asp Asp Pro Lys Ile Thr
Ile Val Ser Cys705 710 715
720Asn Pro Asn Tyr Arg Asn Ser Leu Leu Phe Asn Thr Ser Leu Asp Leu
725 730 735Leu Leu Ser Val Val
Gly Phe Ser Phe Ala Tyr Met Gly Lys Glu Leu 740
745 750Pro Thr Asn Tyr Asn Glu Ala Lys Phe Ile Thr Leu
Ser Met Thr Phe 755 760 765Tyr Phe
Thr Ser Ser Val Ser Leu Cys Thr Phe Met Ser Ala Tyr Ser 770
775 780Gly Val Leu Val Thr Ile Val Asp Leu Leu Val
Thr Val Leu Asn Leu785 790 795
800Leu Ala Ile Ser Leu Gly Tyr Phe Gly Pro Lys Cys Tyr Met Ile Leu
805 810 815Phe Tyr Pro Glu
Arg Asn Thr Pro Ala Tyr Phe Asn Ser Met Ile Gln 820
825 830Gly Tyr Thr Met Arg Arg Asp
8354852PRTHomo sapiens 4Met Leu Gly Pro Ala Val Leu Gly Leu Ser Leu Trp
Ala Leu Leu His1 5 10
15Pro Gly Thr Gly Ala Pro Leu Cys Leu Ser Gln Gln Leu Arg Met Lys
20 25 30Gly Asp Tyr Val Leu Gly Gly
Leu Phe Pro Leu Gly Glu Ala Glu Glu 35 40
45Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Thr
Arg 50 55 60Phe Ser Ser Asn Gly Leu
Leu Trp Ala Leu Ala Met Lys Met Ala Val65 70
75 80Glu Glu Ile Asn Asn Lys Ser Asp Leu Leu Pro
Gly Leu Arg Leu Gly 85 90
95Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Ala Met Lys Pro
100 105 110Ser Leu Met Phe Leu Ala
Lys Ala Gly Ser Arg Asp Ile Ala Ala Tyr 115 120
125Cys Asn Tyr Thr Gln Tyr Gln Pro Arg Val Leu Ala Val Ile
Gly Pro 130 135 140His Ser Ser Glu Leu
Ala Met Val Thr Gly Lys Phe Phe Ser Phe Phe145 150
155 160Leu Met Pro Gln Val Ser Tyr Gly Ala Ser
Met Glu Leu Leu Ser Ala 165 170
175Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val
180 185 190Gln Leu Thr Ala Ala
Ala Glu Leu Leu Gln Glu Phe Gly Trp Asn Trp 195
200 205Val Ala Ala Leu Gly Ser Asp Asp Glu Tyr Gly Arg
Gln Gly Leu Ser 210 215 220Ile Phe Ser
Ala Leu Ala Ala Ala Arg Gly Ile Cys Ile Ala His Glu225
230 235 240Gly Leu Val Pro Leu Pro Arg
Ala Asp Asp Ser Arg Leu Gly Lys Val 245
250 255Gln Asp Val Leu His Gln Val Asn Gln Ser Ser Val
Gln Val Val Leu 260 265 270Leu
Phe Ala Ser Val His Ala Ala His Ala Leu Phe Asn Tyr Ser Ile 275
280 285Ser Ser Arg Leu Ser Pro Lys Val Trp
Val Ala Ser Glu Ala Trp Leu 290 295
300Thr Ser Asp Leu Val Met Gly Leu Pro Gly Met Ala Gln Met Gly Thr305
310 315 320Val Leu Gly Phe
Leu Gln Arg Gly Ala Gln Leu His Glu Phe Pro Gln 325
330 335Tyr Val Lys Thr His Leu Ala Leu Ala Thr
Asp Pro Ala Phe Cys Ser 340 345
350Ala Leu Gly Glu Arg Glu Gln Gly Leu Glu Glu Asp Val Val Gly Gln
355 360 365Arg Cys Pro Gln Cys Asp Cys
Ile Thr Leu Gln Asn Val Ser Ala Gly 370 375
380Leu Asn His His Gln Thr Phe Ser Val Tyr Ala Ala Val Tyr Ser
Val385 390 395 400Ala Gln
Ala Leu His Asn Thr Leu Gln Cys Asn Ala Ser Gly Cys Pro
405 410 415Ala Gln Asp Pro Val Lys Pro
Trp Gln Leu Leu Glu Asn Met Tyr Asn 420 425
430Leu Thr Phe His Val Gly Gly Leu Pro Leu Arg Phe Asp Ser
Ser Gly 435 440 445Asn Val Asp Met
Glu Tyr Asp Leu Lys Leu Trp Val Trp Gln Gly Ser 450
455 460Val Pro Arg Leu His Asp Val Gly Arg Phe Asn Gly
Ser Leu Arg Thr465 470 475
480Glu Arg Leu Lys Ile Arg Trp His Thr Ser Asp Asn Gln Lys Pro Val
485 490 495Ser Arg Cys Ser Arg
Gln Cys Gln Glu Gly Gln Val Arg Arg Val Lys 500
505 510Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp Cys
Glu Ala Gly Ser 515 520 525Tyr Arg
Gln Asn Pro Asp Asp Ile Ala Cys Thr Phe Cys Gly Gln Asp 530
535 540Glu Trp Ser Pro Glu Arg Ser Thr Arg Cys Phe
Arg Arg Arg Ser Arg545 550 555
560Phe Leu Ala Trp Gly Glu Pro Ala Val Leu Leu Leu Leu Leu Leu Leu
565 570 575Ser Leu Ala Leu
Gly Leu Val Leu Ala Ala Leu Gly Leu Phe Val His 580
585 590His Arg Asp Ser Pro Leu Val Gln Ala Ser Gly
Gly Pro Leu Ala Cys 595 600 605Phe
Gly Leu Val Cys Leu Gly Leu Val Cys Leu Ser Val Leu Leu Phe 610
615 620Pro Gly Gln Pro Ser Pro Ala Arg Cys Leu
Ala Gln Gln Pro Leu Ser625 630 635
640His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu Phe Leu Gln Ala
Ala 645 650 655Glu Ile Phe
Val Glu Ser Glu Leu Pro Leu Ser Trp Ala Asp Arg Leu 660
665 670Ser Gly Cys Leu Arg Gly Pro Trp Ala Trp
Leu Val Val Leu Leu Ala 675 680
685Met Leu Val Glu Val Ala Leu Cys Thr Trp Tyr Leu Val Ala Phe Pro 690
695 700Pro Glu Val Val Thr Asp Trp His
Met Leu Pro Thr Glu Ala Leu Val705 710
715 720His Cys Arg Thr Arg Ser Trp Val Ser Phe Gly Leu
Ala His Ala Thr 725 730
735Asn Ala Thr Leu Ala Phe Leu Cys Phe Leu Gly Thr Phe Leu Val Arg
740 745 750Ser Gln Pro Gly Arg Tyr
Asn Arg Ala Arg Gly Leu Thr Phe Ala Met 755 760
765Leu Ala Tyr Phe Ile Thr Trp Val Ser Phe Val Pro Leu Leu
Ala Asn 770 775 780Val Gln Val Val Leu
Arg Pro Ala Val Gln Met Gly Ala Leu Leu Leu785 790
795 800Cys Val Leu Gly Ile Leu Ala Ala Phe His
Leu Pro Arg Cys Tyr Leu 805 810
815Leu Met Arg Gln Pro Gly Leu Asn Thr Pro Glu Phe Phe Leu Gly Gly
820 825 830Gly Pro Gly Asp Ala
Gln Gly Gln Asn Asp Gly Asn Thr Gly Asn Gln 835
840 845Gly Lys His Glu 85052526DNAHomo sapiens
5atgctgctct gcacggctcg cctggtcggc ctgcagcttc tcatttcctg ctgctgggcc
60tttgcctgcc atagcacgga gtcttctcct gacttcaccc tccccggaga ttacctcctg
120gcaggcctgt tccctctcca ttctggctgt ctgcaggtga ggcacagacc cgaggtgacc
180ctgtgtgaca ggtcttgtag cttcaatgag catggctacc acctcttcca ggctatgcgg
240cttggggttg aggagataaa caactccacg gccctgctgc ccaacatcac cctggggtac
300cagctgtatg atgtgtgttc tgactctgcc aatgtgtatg ccacgctgag agtgctctcc
360ctgccagggc aacaccacat agagctccaa ggagaccttc tccactattc ccctacggtg
420ctggcagtga ttgggcctga cagcaccaac cgtgctgcca ccacagccgc cctgctgagc
480cctttcctgg tgcccatgat tagctatgcg gccagcagcg agacgctcag cgtgaagcgg
540cagtatccct ctttcctgcg caccatcccc aatgacaagt accaggtgga gaccatggtg
600ctgctgctgc agaagttcgg gtggacctgg atctctctgg ttggcagcag tgacgactat
660gggcagctag gggtgcaggc actggagaac caggccactg gtcaggggat ctgcattgct
720ttcaaggaca tcatgccctt ctctgcccag gtgggcgatg agaggatgca gtgcctcatg
780cgccacctgg cccaggccgg ggccaccgtc gtggttgttt tttccagccg gcagttggcc
840agggtgtttt tcgagtccgt ggtgctgacc aacctgactg gcaaggtgtg ggtcgcctca
900gaagcctggg ccctctccag gcacatcact ggggtgcccg ggatccagcg cattgggatg
960gtgctgggcg tggccatcca gaagagggct gtccctggcc tgaaggcgtt tgaagaagcc
1020tatgcccggg cagacaagaa ggcccctagg ccttgccaca agggctcctg gtgcagcagc
1080aatcagctct gcagagaatg ccaagctttc atggcacaca cgatgcccaa gctcaaagcc
1140ttctccatga gttctgccta caacgcatac cgggctgtgt atgcggtggc ccatggcctc
1200caccagctcc tgggctgtgc ctctggagct tgttccaggg gccgagtcta cccctggcag
1260cttttggagc agatccacaa ggtgcatttc cttctacaca aggacactgt ggcgtttaat
1320gacaacagag atcccctcag tagctataac ataattgcct gggactggaa tggacccaag
1380tggaccttca cggtcctcgg ttcctccaca tggtctccag ttcagctaaa cataaatgag
1440accaaaatcc agtggcacgg aaaggacaac caggtgccta agtctgtgtg ttccagcgac
1500tgtcttgaag ggcaccagcg agtggttacg ggtttccatc actgctgctt tgagtgtgtg
1560ccctgtgggg ctgggacctt cctcaacaag agtgacctct acagatgcca gccttgtggg
1620aaagaagagt gggcacctga gggaagccag acctgcttcc cgcgcactgt ggtgtttttg
1680gctttgcgtg agcacacctc ttgggtgctg ctggcagcta acacgctgct gctgctgctg
1740ctgcttggga ctgctggcct gtttgcctgg cacctagaca cccctgtggt gaggtcagca
1800gggggccgcc tgtgctttct tatgctgggc tccctggcag caggtagtgg cagcctctat
1860ggcttctttg gggaacccac aaggcctgcg tgcttgctac gccaggccct ctttgccctt
1920ggtttcacca tcttcctgtc ctgcctgaca gttcgctcat tccaactaat catcatcttc
1980aagttttcca ccaaggtacc tacattctac cacgcctggg tccaaaacca cggtgctggc
2040ctgtttgtga tgatcagctc agcggcccag ctgcttatct gtctaacttg gctggtggtg
2100tggaccccac tgcctgctag ggaataccag cgcttccccc atctggtgat gcttgagtgc
2160acagagacca actccctggg cttcatactg gccttcctct acaatggcct cctctccatc
2220agtgcctttg cctgcagcta cctgggtaag gacttgccag agaactacaa cgaggccaaa
2280tgtgtcacct tcagcctgct cttcaacttc gtgtcctgga tcgccttctt caccacggcc
2340agcgtctacg acggcaagta cctgcctgcg gccaacatga tggctgggct gagcagcctg
2400agcagcggct tcggtgggta ttttctgcct aagtgctacg tgatcctctg ccgcccagac
2460ctcaacagca cagagcactt ccaggcctcc attcaggact acacgaggcg ctgcggctcc
2520acctga
252662559DNAHomo sapiens 6atgctgggcc ctgctgtcct gggcctcagc ctctgggctc
tcctgcaccc tgggacgggg 60gccccattgt gcctgtcaca gcaacttagg atgaaggggg
actacgtgct gggggggctg 120ttccccctgg gcgaggccga ggaggctggc ctccgcagcc
ggacacggcc cagcagccct 180gtgtgcacca ggttctcctc aaacggcctg ctctgggcac
tggccatgaa aatggccgtg 240gaggagatca acaacaagtc ggatctgctg cccgggctgc
gcctgggcta cgacctcttt 300gatacgtgct cggagcctgt ggtggccatg aagcccagcc
tcatgttcct ggccaaggca 360ggcagccgcg acatcgccgc ctactgcaac tacacgcagt
accagccccg tgtgctggct 420gtcatcgggc cccactcgtc agagctcgcc atggtcaccg
gcaagttctt cagcttcttc 480ctcatgcccc aggtcagcta cggtgctagc atggagctgc
tgagcgcccg ggagaccttc 540ccctccttct tccgcaccgt gcccagcgac cgtgtgcagc
tgacggccgc cgcggagctg 600ctgcaggagt tcggctggaa ctgggtggcc gccctgggca
gcgacgacga gtacggccgg 660cagggcctga gcatcttctc ggccctggcc gcggcacgcg
gcatctgcat cgcgcacgag 720ggcctggtgc cgctgccccg tgccgatgac tcgcggctgg
ggaaggtgca ggacgtcctg 780caccaggtga accagagcag cgtgcaggtg gtgctgctgt
tcgcctccgt gcacgccgcc 840cacgccctct tcaactacag catcagcagc aggctctcgc
ccaaggtgtg ggtggccagc 900gaggcctggc tgacctctga cctggtcatg gggctgcccg
gcatggccca gatgggcacg 960gtgcttggct tcctccagag gggtgcccag ctgcacgagt
tcccccagta cgtgaagacg 1020cacctggccc tggccaccga cccggccttc tgctctgccc
tgggcgagag ggagcagggt 1080ctggaggagg acgtggtggg ccagcgctgc ccgcagtgtg
actgcatcac gctgcagaac 1140gtgagcgcag ggctaaatca ccaccagacg ttctctgtct
acgcagctgt gtatagcgtg 1200gcccaggccc tgcacaacac tcttcagtgc aacgcctcag
gctgccccgc gcaggacccc 1260gtgaagccct ggcagctcct ggagaacatg tacaacctga
ccttccacgt gggcgggctg 1320ccgctgcggt tcgacagcag cggaaacgtg gacatggagt
acgacctgaa gctgtgggtg 1380tggcagggct cagtgcccag gctccacgac gtgggcaggt
tcaacggcag cctcaggaca 1440gagcgcctga agatccgctg gcacacgtct gacaaccaga
agcccgtgtc ccggtgctcg 1500cggcagtgcc aggagggcca ggtgcgccgg gtcaaggggt
tccactcctg ctgctacgac 1560tgtgtggact gcgaggcggg cagctaccgg caaaacccag
acgacatcgc ctgcaccttt 1620tgtggccagg atgagtggtc cccggagcga agcacacgct
gcttccgccg caggtctcgg 1680ttcctggcat ggggcgagcc ggctgtgctg ctgctgctcc
tgctgctgag cctggcgctg 1740ggccttgtgc tggctgcttt ggggctgttc gttcaccatc
gggacagccc actggttcag 1800gcctcggggg ggcccctggc ctgctttggc ctggtgtgcc
tgggcctggt ctgcctcagc 1860gtcctcctgt tccctggcca gcccagccct gcccgatgcc
tggcccagca gcccttgtcc 1920cacctcccgc tcacgggctg cctgagcaca ctcttcctgc
aggcggccga gatcttcgtg 1980gagtcagaac tgcctctgag ctgggcagac cggctgagtg
gctgcctgcg ggggccctgg 2040gcctggctgg tggtgctgct ggccatgctg gtggaggtcg
cactgtgcac ctggtacctg 2100gtggccttcc cgccggaggt ggtgacggac tggcacatgc
tgcccacgga ggcgctggtg 2160cactgccgca cacgctcctg ggtcagcttc ggcctagcgc
acgccaccaa tgccacgctg 2220gcctttctct gcttcctggg cactttcctg gtgcggagcc
agccgggctg ctacaaccgt 2280gcccgtggcc tcacctttgc catgctggcc tacttcatca
cctgggtctc ctttgtgccc 2340ctcctggcca atgtgcaggt ggtcctcagg cccgccgtgc
agatgggcgc cctcctgctc 2400tgtgtcctgg gcatcctggc tgccttccac ctgcccaggt
gttacctgct catgcggcag 2460ccagggctca acacccccga gttcttcctg ggagggggcc
ctggggatgc ccaaggccag 2520aatgacggga acacaggaaa tcaggggaaa catgagtga
255972519DNAHomo sapiens 7atggggccca gggcaaagac
catctgctcc ctgttcttcc tcctatgggt cctggctgag 60ccggctgaga actcggactt
ctacctgcct ggggattacc tcctgggtgg cctcttctcc 120ctccatgcca acatgaaggg
cattgttcac cttaacttcc tgcaggtgcc catgtgcaag 180gagtatgaag tgaaggtgat
aggctacaac ctcatgcagg ccatgcgctt cgcggtggag 240gagatcaaca atgacagcag
cctgctgcct ggtgtgctgc tgggctatga gatcgtggat 300gtgtgctaca tctccaacaa
tgtccagccg gtgctctact tcctggcaca cgaggacaac 360ctccttccca tccaagagga
ctacagtaac tacatttccc gtgtggtggc tgtcattggc 420cctgacaact ccgagtctgt
catgactgtg gccaacttcc tctccctatt tctccttcca 480cagatcacct acagcgccat
cagcgatgag ctgcgagaca aggtgcgctt cccggctttg 540ctgcgtacca cacccagcgc
cgaccaccac gtcgaggcca tggtgcagct gatgctgcac 600ttccgctgga actggatcat
tgtgctggtg agcagcgaca cctatggccg cgacaatggc 660agctgcttgg cgagcgcgtg
gcccggcgcg acatctgcat cgccttccag gagacgctgc 720ccacactgca gcccaaccag
aacatgacgt cagaggagcg ccagcgcctg gtgaccattg 780tggacaagct gcagcagagc
acagcgcgcg tcgtggtcgt gttctcgccc gacctgaccc 840tgtaccactt cttcaatgag
gtgctgcgcc agaacttcac gggcgccgtg tggatcgcct 900ccgagtcctg ggccatcgac
ccggtcctgc acaacctcac ggagctgggc cacttgggca 960ccttcctggg catcaccatc
cagagcgtgc ccatcccggg cttcagtgag ttccgcgagt 1020ggggcccaca ggctgggccg
ccacccctca gcaggaccag ccagagctat acctgcaacc 1080aggagtgcga caactgcctg
aacgccacct tgtccttcaa caccattctc aggctctctg 1140gggagcgtgt cgtctacagc
gtgtactctg cggtctatgc tgtggcccat gccctgcaca 1200gcctcctcgg ctgtgacaaa
agcacctgca ccaagagggt ggtctacccc tggcagctgc 1260ttgaggagat ctggaaggtc
aacttcactc tcctggacca ccaaatcttc ttcgacccgc 1320aaggggacgt ggctctgcac
ttggagattg tccagtggca atgggaccgg agccagaatc 1380ccttccagag cgtcgcctcc
tactaccccc tgcagcgaca gctgaagaac atccaagaca 1440tctcctggca caccgtcaac
aacacgatcc ctatgtccat gtgttccaag aggtgccagt 1500cagggcaaaa gaagaagcct
gtgggcatcc acgtctgctg cttcgagtgc atcgactgcc 1560ttcccggcac cttcctcaac
cacactgaag atgaatatga atgccaggcc tgcccgaata 1620acgagtggtc ctaccagagt
gagacctcct gcttcaagcg gcagctggtc ttcctggaat 1680ggcatgaggc acccaccatc
gctgtggccc tgctggccgc cctgggcttc ctcagcaccc 1740tggccatcct ggtgatattc
tggaggcact tccagacacc catagttcgc tcggctgggg 1800gccccatgtg cttcctgatg
ctgacactgc tgctggtggc atacatggtg gtcccggtgt 1860acgtggggcc gcccaaggtc
tccacctgcc tctgccgcca ggccctcttt cccctctgct 1920tcacaatttg catctcctgt
atcgccgtgc gttctttcca gatcgtctgc gccttcaaga 1980tggccagccg cttcccacgc
gcctacagct actgggtccg ctaccagggg ccctacgtct 2040ctatggcatt tatcacggta
ctcaaaatgg tcattgtggt aattcgcatg ctggccacgg 2100gcctcagtcc caccacccgt
actgaccccg atgaccccaa gatcacaatt gtctcctgta 2160accccaacta ccgcaacagc
ctgctgttca acaccagcct ggacctgctg ctctcagtgg 2220tgggtttcag cttcgcctac
atgggcaaag agctgcccac caactacaac gaggccaagt 2280tcatcaccct cagcatgacc
ttctatttca cctcatccgt ctccctctgc accttcatgt 2340ctgcctacag cggggtgctg
gtcaccatcg tggacctctt ggtcactgtg ctcaacctcc 2400tggccatcag cctgggctac
ttcggcccca agtgctacat gatcctcttc tacccggagc 2460gcaacacgcc cgcctacttc
aacagcatga tccagggcta caccatgagg agggactag 251982577DNAHomo sapiens
8atgccgggtt tggctatctt gggcctcagt ctggctgctt tcctggagct tgggatgggg
60tcctctttgt gtctgtcaca gcaattcaag gcacaagggg actatatatt gggtggacta
120tttcccctgg gcacaactga ggaggccact ctcaaccaga gaacacagcc caacggcatc
180ctatgtacca ggttctcgcc ccttggtttg ttcctggcca tggctatgaa gatggctgta
240gaggagatca acaatggatc tgccttgctc cctgggctgc gactgggcta tgacctgttt
300gacacatgct cagagccagt ggtcaccatg aagcccagcc tcatgttcat ggccaaggtg
360ggaagtcaaa gcattgctgc ctactgcaac tacacacagt accaaccccg tgtgctggct
420gtcattggtc cccactcatc agagcttgcc ctcattacag gcaagttctt cagcttcttc
480ctcatgccac aggtcagcta tagtgccagc atggatcggc taagtgaccg ggaaacattt
540ccatccttct tccgcacagt gcccagtgac cgggtgcagc tgcaggccgt tgtgacactg
600ttgcagaatt tcagctggaa ctgggtggct gccttaggta gtgatgatga ctatggccgg
660gaaggtctga gcatcttttc tggtctggcc aactcacgag gtatctgcat tgcacacgag
720ggcctggtgc cacaacatga cactagtggc caacaattgg gcaaggtggt ggatgtgcta
780cgccaagtga accaaagcaa agtacaggtg gtggtgctgt ttgcatctgc ccgtgctgtc
840tactcccttt ttagctacag catccttcat gacctctcac ccaaggtatg ggtggccagt
900gagtcctggc tgacctctga cctggtcatg acacttccca atattgcccg tgtgggcact
960gttcttgggt ttctgcagcg cggtgcccta ctgcctgaat tttcccatta tgtggagact
1020cgccttgccc tagctgctga cccaacattc tgtgcctccc tgaaagctga gttggatctg
1080gaggagcgcg tgatggggcc acgctgttca caatgtgact acatcatgct acagaacctg
1140tcatctgggc tgatgcagaa cctatcagct gggcagttgc accaccaaat atttgcaacc
1200tatgcagctg tgtacagtgt ggctcaggcc cttcacaaca ccctgcagtg caatgtctca
1260cattgccaca catcagagcc tgttcaaccc tggcagctcc tggagaacat gtacaatatg
1320agtttccgtg ctcgagactt gacactgcag tttgatgcca aagggagtgt agacatggaa
1380tatgacctga agatgtgggt gtggcagagc cctacacctg tactacatac tgtaggcacc
1440ttcaacggca cccttcagct gcagcactcg aaaatgtatt ggccaggcaa ccaggtgcca
1500gtctcccagt gctcccggca gtgcaaagat ggccaggtgc gcagagtaaa gggctttcat
1560tcctgctgct atgactgtgt ggactgcaag gcagggagct accggaagca tccagatgac
1620ttcacctgta ctccatgtgg caaggatcag tggtccccag aaaaaagcac aacctgctta
1680cctcgcaggc ccaagtttct ggcttggggg gagccagctg tgctgtcact tctcctgctg
1740ctttgcctgg tgctgggcct gacactggct gccctggggc tctttgtcca ctactgggac
1800agccctcttg ttcaggcctc aggtgggtca ctgttctgct ttggcctgat ctgcctaggc
1860ctcttctgcc tcagtgtcct tctgttccca ggacgaccac gctctgccag ctgccttgcc
1920caacaaccaa tggctcacct ccctctcaca ggctgcctga gcacactctt cctgcaagca
1980gccgagatct ttgtggagtc tgagctgcca ctgagttggg caaactggct ctgcagctac
2040cttcggggcc cctgggcttg gctggtggta ctgctggcca ctcttgtgga ggctgcacta
2100tgtgcctggt acttgatggc tttccctcca gaggtggtga cagattggca ggtgctgccc
2160acggaggtac tggaacactg ccgcatgcgt tcctgggtca gcctgggctt ggtgcacatc
2220accaatgcag tgttagcttt cctctgcttt ctgggcactt tcctggtaca gagccagcct
2280ggtcgctata accgtgcccg tggcctcacc ttcgccatgc tagcttattt catcatctgg
2340gtctcttttg tgcccctcct ggctaatgtg caggtggcct accagccagc tgtgcagatg
2400ggtgctatct tattctgtgc cctgggcatc ctggccacct tccacctgcc caaatgctat
2460gtacttctgt ggctgccaga gctcaacacc caggagttct tcctgggaag gagccccaag
2520gaagcatcag atgggaatag tggtagtagt gaggcaactc ggggacacag tgaatga
25779842PRTMus musculus 9Met Leu Phe Trp Ala Ala His Leu Leu Leu Ser Leu
Gln Leu Ala Val1 5 10
15Ala Tyr Cys Trp Ala Phe Ser Cys Gln Arg Thr Glu Ser Ser Pro Gly
20 25 30Phe Ser Leu Pro Gly Asp Phe
Leu Leu Ala Gly Leu Phe Ser Leu His 35 40
45Ala Asp Cys Leu Gln Val Arg His Arg Pro Leu Val Thr Ser Cys
Asp 50 55 60Arg Ser Asp Ser Phe Asn
Gly His Gly Tyr His Leu Phe Gln Ala Met65 70
75 80Arg Phe Thr Val Glu Glu Ile Asn Asn Ser Thr
Ala Leu Leu Pro Asn 85 90
95Ile Thr Leu Gly Tyr Glu Leu Tyr Asp Val Cys Ser Glu Ser Ser Asn
100 105 110Val Tyr Ala Thr Leu Arg
Val Leu Ala Gln Gln Gly Thr Gly His Leu 115 120
125Glu Met Gln Arg Asp Leu Arg Asn His Ser Ser Lys Val Val
Ala Leu 130 135 140Ile Gly Pro Asp Asn
Thr Asp His Ala Val Thr Thr Ala Ala Leu Leu145 150
155 160Ser Pro Phe Leu Met Pro Leu Val Ser Tyr
Glu Ala Ser Ser Val Ile 165 170
175Leu Ser Gly Lys Arg Lys Phe Pro Ser Phe Leu Arg Thr Ile Pro Ser
180 185 190Asp Lys Tyr Gln Val
Glu Val Ile Val Arg Leu Leu Gln Ser Phe Gly 195
200 205Trp Val Trp Ile Ser Leu Val Gly Ser Tyr Gly Asp
Tyr Gly Gln Leu 210 215 220Gly Val Gln
Ala Leu Glu Glu Leu Ala Thr Pro Arg Gly Ile Cys Val225
230 235 240Ala Phe Lys Asp Val Val Pro
Leu Ser Ala Gln Ala Gly Asp Pro Arg 245
250 255Met Gln Arg Met Met Leu Arg Leu Ala Arg Ala Arg
Thr Thr Val Val 260 265 270Val
Val Phe Ser Asn Arg His Leu Ala Gly Val Phe Phe Arg Ser Val 275
280 285Val Leu Ala Asn Leu Thr Gly Lys Val
Trp Ile Ala Ser Glu Asp Trp 290 295
300Ala Ile Ser Thr Tyr Ile Thr Asn Val Pro Gly Ile Gln Gly Ile Gly305
310 315 320Thr Val Leu Gly
Val Ala Ile Gln Gln Arg Gln Val Pro Gly Leu Lys 325
330 335Glu Phe Glu Glu Ser Tyr Val Gln Ala Val
Met Gly Ala Pro Arg Thr 340 345
350Cys Pro Glu Gly Ser Trp Cys Gly Thr Asn Gln Leu Cys Arg Glu Cys
355 360 365His Ala Phe Thr Thr Trp Asn
Met Pro Glu Leu Gly Ala Phe Ser Met 370 375
380Ser Ala Ala Tyr Asn Val Tyr Glu Ala Val Tyr Ala Val Ala His
Gly385 390 395 400Leu His
Gln Leu Leu Gly Cys Thr Ser Gly Thr Cys Ala Arg Gly Pro
405 410 415Val Tyr Pro Trp Gln Leu Leu
Gln Gln Ile Tyr Lys Val Asn Phe Leu 420 425
430Leu His Lys Lys Thr Val Ala Phe Asp Asp Lys Gly Asp Pro
Leu Gly 435 440 445Tyr Tyr Asp Ile
Ile Ala Trp Asp Trp Asn Gly Pro Glu Trp Thr Phe 450
455 460Glu Val Ile Gly Ser Ala Ser Leu Ser Pro Val His
Leu Asp Ile Asn465 470 475
480Lys Thr Lys Ile Gln Trp His Gly Lys Asn Asn Gln Val Pro Val Ser
485 490 495Val Cys Thr Arg Asp
Cys Leu Glu Gly His His Arg Leu Val Met Gly 500
505 510Ser His His Cys Cys Phe Glu Cys Met Pro Cys Glu
Ala Gly Thr Phe 515 520 525Leu Asn
Thr Ser Glu Leu His Thr Cys Gln Pro Cys Gly Thr Glu Glu 530
535 540Trp Ala Pro Glu Gly Ser Ser Ala Cys Phe Ser
Arg Thr Val Glu Phe545 550 555
560Leu Gly Trp His Glu Pro Ile Ser Leu Val Leu Leu Ala Ala Asn Thr
565 570 575Leu Leu Leu Leu
Leu Leu Ile Gly Thr Ala Gly Leu Phe Ala Trp Arg 580
585 590Leu His Thr Pro Val Val Arg Ser Ala Gly Gly
Arg Leu Cys Phe Leu 595 600 605Met
Leu Gly Ser Leu Val Ala Gly Ser Cys Ser Leu Tyr Ser Phe Phe 610
615 620Gly Lys Pro Thr Val Pro Ala Cys Leu Leu
Arg Gln Pro Leu Phe Ser625 630 635
640Leu Gly Phe Ala Ile Phe Leu Ser Cys Leu Thr Ile Arg Ser Phe
Gln 645 650 655Leu Val Ile
Ile Phe Lys Phe Ser Thr Lys Val Pro Thr Phe Tyr His 660
665 670Thr Trp Ala Gln Asn His Gly Ala Gly Ile
Phe Val Ile Val Ser Ser 675 680
685Thr Val His Leu Phe Leu Cys Leu Thr Trp Leu Ala Met Trp Thr Pro 690
695 700Arg Pro Thr Arg Glu Tyr Gln Arg
Phe Pro His Leu Val Ile Leu Glu705 710
715 720Cys Thr Glu Val Asn Ser Val Gly Phe Leu Val Ala
Phe Ala His Asn 725 730
735Ile Leu Leu Ser Ile Ser Thr Phe Val Cys Ser Tyr Leu Gly Lys Glu
740 745 750Leu Pro Glu Asn Tyr Asn
Glu Ala Lys Cys Val Thr Phe Ser Leu Leu 755 760
765Leu His Phe Val Ser Trp Ile Ala Phe Phe Thr Met Ser Ser
Ile Tyr 770 775 780Gln Gly Ser Tyr Leu
Pro Ala Val Asn Val Leu Ala Gly Leu Ala Thr785 790
795 800Leu Ser Gly Gly Phe Ser Gly Tyr Phe Leu
Pro Lys Cys Tyr Val Ile 805 810
815Leu Cys Arg Pro Glu Leu Asn Asn Thr Glu His Phe Gln Ala Ser Ile
820 825 830Gln Asp Tyr Thr Arg
Arg Cys Gly Thr Thr 835 84010842PRTMus musculus
10Met Leu Phe Trp Ala Ala His Leu Leu Leu Ser Leu Gln Leu Ala Val1
5 10 15Ala Tyr Cys Trp Ala Phe
Ser Cys Gln Arg Thr Glu Ser Ser Pro Gly 20 25
30Phe Ser Leu Pro Gly Asp Phe Leu Leu Ala Gly Leu Phe
Ser Leu His 35 40 45Ala Asp Cys
Leu Gln Val Arg His Arg Pro Leu Val Thr Ser Cys Asp 50
55 60Arg Ser Asp Ser Phe Asn Gly His Gly Tyr His Leu
Phe Gln Ala Met65 70 75
80Arg Phe Thr Val Glu Glu Ile Asn Asn Ser Thr Ala Leu Leu Pro Asn
85 90 95Ile Thr Leu Gly Tyr Glu
Leu Tyr Asp Val Cys Ser Glu Ser Ser Asn 100
105 110Val Tyr Ala Thr Leu Arg Val Leu Ala Gln Gln Gly
Thr Gly His Leu 115 120 125Glu Met
Gln Arg Asp Leu Arg Asn His Ser Ser Lys Val Val Ala Leu 130
135 140Ile Gly Pro Asp Asn Thr Asp His Ala Val Thr
Thr Ala Ala Leu Leu145 150 155
160Ser Pro Phe Leu Met Pro Leu Val Ser Tyr Glu Ala Ser Ser Val Ile
165 170 175Leu Ser Gly Lys
Arg Lys Phe Pro Ser Phe Leu Arg Thr Ile Pro Ser 180
185 190Asp Lys Tyr Gln Val Glu Val Ile Val Arg Leu
Leu Gln Ser Phe Gly 195 200 205Trp
Val Trp Ile Ser Leu Val Gly Ser Tyr Gly Asp Tyr Gly Gln Leu 210
215 220Gly Val Gln Ala Leu Glu Glu Leu Ala Thr
Pro Arg Gly Ile Cys Val225 230 235
240Ala Phe Lys Asp Val Val Pro Leu Ser Ala Gln Ala Gly Asp Pro
Arg 245 250 255Met Gln Arg
Met Met Leu Arg Leu Ala Arg Ala Arg Thr Thr Val Val 260
265 270Val Val Phe Ser Asn Arg His Leu Ala Gly
Val Phe Phe Arg Ser Val 275 280
285Val Leu Ala Asn Leu Thr Gly Lys Val Trp Ile Ala Ser Glu Asp Trp 290
295 300Ala Ile Ser Thr Tyr Ile Thr Asn
Val Pro Gly Ile Gln Gly Ile Gly305 310
315 320Thr Val Leu Gly Val Ala Ile Gln Gln Arg Gln Val
Pro Gly Leu Lys 325 330
335Glu Phe Glu Glu Ser Tyr Val Gln Ala Val Met Gly Ala Pro Arg Thr
340 345 350Cys Pro Glu Gly Ser Trp
Cys Gly Thr Asn Gln Leu Cys Arg Glu Cys 355 360
365His Ala Phe Thr Thr Trp Asn Met Pro Glu Leu Gly Ala Phe
Ser Met 370 375 380Ser Ala Ala Tyr Asn
Val Tyr Glu Ala Val Tyr Ala Val Ala His Gly385 390
395 400Leu His Gln Leu Leu Gly Cys Thr Ser Gly
Thr Cys Ala Arg Gly Pro 405 410
415Val Tyr Pro Trp Gln Leu Leu Gln Gln Ile Tyr Lys Val Asn Phe Leu
420 425 430Leu His Lys Lys Thr
Val Ala Phe Asp Asp Lys Gly Asp Pro Leu Gly 435
440 445Tyr Tyr Asp Ile Ile Ala Trp Asp Trp Asn Gly Pro
Glu Trp Thr Phe 450 455 460Glu Val Ile
Gly Ser Ala Ser Leu Ser Pro Val His Leu Asp Ile Asn465
470 475 480Lys Thr Lys Ile Gln Trp His
Gly Lys Asn Asn Gln Val Pro Val Ser 485
490 495Val Cys Thr Arg Asp Cys Leu Glu Gly His His Arg
Leu Val Met Gly 500 505 510Ser
His His Cys Cys Phe Glu Cys Met Pro Cys Glu Ala Gly Thr Phe 515
520 525Leu Asn Thr Ser Glu Leu His Thr Cys
Gln Pro Cys Gly Thr Glu Glu 530 535
540Trp Ala Pro Glu Gly Ser Ser Ala Cys Phe Ser Arg Thr Val Glu Phe545
550 555 560Leu Gly Trp His
Glu Pro Ile Ser Leu Val Leu Leu Ala Ala Asn Thr 565
570 575Leu Leu Leu Leu Leu Leu Ile Gly Thr Ala
Gly Leu Phe Ala Trp Arg 580 585
590Leu His Thr Pro Val Val Arg Ser Ala Gly Gly Arg Leu Cys Phe Leu
595 600 605Met Leu Gly Ser Leu Val Ala
Gly Ser Cys Ser Leu Tyr Ser Phe Phe 610 615
620Gly Lys Pro Thr Val Pro Ala Cys Leu Leu Arg Gln Pro Leu Phe
Ser625 630 635 640Leu Gly
Phe Ala Ile Phe Leu Ser Cys Leu Thr Ile Arg Ser Phe Gln
645 650 655Leu Val Ile Ile Phe Lys Phe
Ser Thr Lys Val Pro Thr Phe Tyr His 660 665
670Thr Trp Ala Gln Asn His Gly Ala Gly Ile Phe Val Ile Val
Ser Ser 675 680 685Thr Val His Leu
Phe Leu Cys Leu Thr Trp Leu Ala Met Trp Thr Pro 690
695 700Arg Pro Thr Arg Glu Tyr Gln Arg Phe Pro His Leu
Val Ile Leu Glu705 710 715
720Cys Thr Glu Val Asn Ser Val Gly Phe Leu Val Ala Phe Ala His Asn
725 730 735Ile Leu Leu Ser Ile
Ser Thr Phe Val Cys Ser Tyr Leu Gly Lys Glu 740
745 750Leu Pro Glu Asn Tyr Asn Glu Ala Lys Cys Val Thr
Phe Ser Leu Leu 755 760 765Leu His
Phe Val Ser Trp Ile Ala Phe Phe Thr Met Ser Ser Ile Tyr 770
775 780Gln Gly Ser Tyr Leu Pro Ala Val Asn Val Leu
Ala Gly Leu Ala Thr785 790 795
800Leu Ser Gly Gly Phe Ser Gly Tyr Phe Leu Pro Lys Cys Tyr Val Ile
805 810 815Leu Cys Arg Pro
Glu Leu Asn Asn Thr Glu His Phe Gln Ala Ser Ile 820
825 830Gln Asp Tyr Thr Arg Arg Cys Gly Thr Thr
835 84011842PRTHomo sapiens 11Met Leu Phe Trp Ala Ala
His Leu Leu Leu Ser Leu Gln Leu Ala Val1 5
10 15Ala Tyr Cys Trp Ala Phe Ser Cys Gln Arg Thr Glu
Ser Ser Pro Gly 20 25 30Phe
Ser Leu Pro Gly Asp Phe Leu Leu Ala Gly Leu Phe Ser Leu His 35
40 45Ala Asp Cys Leu Gln Val Arg His Arg
Pro Leu Val Thr Ser Cys Asp 50 55
60Arg Ser Asp Ser Phe Asn Gly His Gly Tyr His Leu Phe Gln Ala Met65
70 75 80Arg Phe Thr Val Glu
Glu Ile Asn Asn Ser Thr Ala Leu Leu Pro Asn 85
90 95Ile Thr Leu Gly Tyr Glu Leu Tyr Asp Val Cys
Ser Glu Ser Ser Asn 100 105
110Val Tyr Ala Thr Leu Arg Val Leu Ala Gln Gln Gly Thr Gly His Leu
115 120 125Glu Met Gln Arg Asp Leu Arg
Asn His Ser Ser Lys Val Val Ala Leu 130 135
140Ile Gly Pro Asp Asn Thr Asp His Ala Val Thr Thr Ala Ala Leu
Leu145 150 155 160Ser Pro
Phe Leu Met Pro Leu Val Ser Tyr Glu Ala Ser Ser Val Ile
165 170 175Leu Ser Gly Lys Arg Lys Phe
Pro Ser Phe Leu Arg Thr Ile Pro Ser 180 185
190Asp Lys Tyr Gln Val Glu Val Ile Val Arg Leu Leu Gln Ser
Phe Gly 195 200 205Trp Val Trp Ile
Ser Leu Val Gly Ser Tyr Gly Asp Tyr Gly Gln Leu 210
215 220Gly Val Gln Ala Leu Glu Glu Leu Ala Thr Pro Arg
Gly Ile Cys Val225 230 235
240Ala Phe Lys Asn Val Val Pro Leu Ser Ala Gln Ala Gly Asp Pro Arg
245 250 255Met Gln Arg Met Met
Leu Arg Leu Ala Arg Ala Arg Thr Thr Val Val 260
265 270Val Val Phe Ser Asn Arg His Leu Asp Gly Val Phe
Phe Arg Ser Val 275 280 285Val Leu
Ala Asn Leu Thr Gly Lys Val Trp Ile Ala Ser Glu Asp Trp 290
295 300Ala Ile Ser Thr Asn Ile Pro Asn Val Ser Gly
Ile Gln Gly Ile Gly305 310 315
320Thr Val Leu Gly Val Ala Ile Gln Gln Arg Gln Val Pro Gly Leu Lys
325 330 335Glu Phe Glu Glu
Ser Tyr Val Gln Ala Val Met Gly Ala Pro Arg Thr 340
345 350Cys Pro Glu Gly Ser Trp Cys Gly Thr Asn Gln
Leu Cys Arg Glu Cys 355 360 365His
Ala Phe Thr Thr Trp Asn Met Pro Glu Leu Gly Ala Phe Ser Met 370
375 380Ser Ala Ala Tyr Asn Val Tyr Glu Ala Val
Tyr Ala Val Ala His Gly385 390 395
400Leu His Gln Leu Leu Gly Cys Thr Ser Gly Thr Cys Ala Arg Gly
Pro 405 410 415Val His Pro
Trp Gln Leu Leu Gln Gln Ile Tyr Lys Val Asn Phe Leu 420
425 430Leu His Lys Lys Thr Val Ala Phe Asp Asp
Lys Gly Asp Pro Leu Gly 435 440
445Tyr Tyr Asp Ile Ile Ala Trp Asp Trp Asn Gly Pro Glu Trp Thr Phe 450
455 460Glu Val Ile Gly Ser Ala Ser Leu
Ser Pro Val His Leu Asp Ile Asn465 470
475 480Lys Thr Lys Ile Gln Trp His Gly Lys Asn Asn Gln
Val Pro Val Ser 485 490
495Val Cys Thr Arg Asp Cys Leu Glu Gly His His Arg Leu Val Met Gly
500 505 510Ser His His Cys Cys Phe
Glu Cys Met Pro Cys Glu Ala Gly Thr Phe 515 520
525Leu Asn Thr Ser Glu Leu His Thr Cys Gln Pro Cys Gly Thr
Glu Glu 530 535 540Trp Ala Pro Glu Gly
Ser Ser Ala Cys Phe Ser Arg Thr Val Glu Phe545 550
555 560Leu Gly Trp His Glu Pro Ile Ser Leu Val
Leu Leu Ala Ala Asn Thr 565 570
575Leu Leu Leu Leu Leu Leu Ile Gly Thr Ala Gly Leu Phe Ala Trp Arg
580 585 590Leu His Thr Pro Val
Val Arg Ser Ala Gly Gly Arg Leu Cys Phe Leu 595
600 605Met Leu Gly Ser Leu Val Ala Gly Ser Cys Ser Leu
Tyr Ser Phe Phe 610 615 620Gly Lys Pro
Thr Val Pro Ala Cys Leu Leu Arg Gln Pro Leu Phe Ser625
630 635 640Leu Gly Phe Ala Ile Phe Leu
Ser Cys Leu Thr Ile Arg Ser Phe Gln 645
650 655Leu Val Ile Ile Phe Lys Phe Ser Thr Lys Val Pro
Thr Phe Tyr His 660 665 670Thr
Trp Ala Gln Asn His Gly Ala Gly Ile Phe Val Ile Val Ser Ser 675
680 685Thr Val His Leu Phe Leu Cys Leu Thr
Trp Leu Ala Met Trp Thr Pro 690 695
700Arg Pro Thr Arg Glu Tyr Gln Arg Phe Pro His Leu Val Ile Leu Glu705
710 715 720Cys Thr Glu Val
Asn Ser Val Gly Phe Leu Val Ala Phe Ala His Asn 725
730 735Ile Leu Leu Ser Ile Ser Thr Phe Val Cys
Ser Tyr Leu Gly Lys Glu 740 745
750Leu Pro Glu Asn Tyr Asn Glu Ala Lys Cys Val Thr Phe Ser Leu Leu
755 760 765Leu His Phe Val Ser Trp Ile
Ala Phe Phe Thr Met Ser Ser Ile Tyr 770 775
780Gln Gly Ser Tyr Leu Pro Ala Val Asn Val Leu Ala Gly Leu Ala
Thr785 790 795 800Leu Ser
Gly Gly Phe Ser Gly Tyr Phe Leu Pro Lys Cys Tyr Val Ile
805 810 815Leu Cys Arg Pro Glu Leu Asn
Asn Thr Glu His Phe Gln Ala Ser Ile 820 825
830Gln Asp Tyr Thr Arg Arg Cys Gly Thr Thr 835
84012841PRTHomo sapiens 12Met Leu Leu Cys Thr Ala Arg Leu Val
Gly Leu Gln Leu Leu Ile Ser1 5 10
15Cys Cys Trp Ala Phe Ala Cys His Ser Thr Glu Ser Ser Pro Asp
Phe 20 25 30Thr Leu Pro Gly
Asp Tyr Leu Leu Ala Gly Leu Phe Pro Leu His Ser 35
40 45Gly Cys Leu Gln Val Arg His Arg Pro Glu Val Thr
Leu Cys Asp Arg 50 55 60Ser Cys Ser
Phe Asn Glu His Gly Tyr His Leu Phe Gln Ala Met Arg65 70
75 80Leu Gly Val Glu Glu Ile Asn Asn
Ser Thr Ala Leu Leu Pro Asn Ile 85 90
95Thr Leu Gly Tyr Gln Leu Tyr Asp Val Cys Ser Asp Ser Ala
Asn Val 100 105 110Tyr Ala Thr
Leu Arg Val Leu Ser Leu Pro Gly Gln His His Ile Glu 115
120 125Leu Gln Gly Asp Leu Leu His Tyr Ser Pro Thr
Val Leu Ala Val Ile 130 135 140Gly Pro
Asp Ser Thr Asn Arg Ala Ala Thr Thr Ala Ala Leu Leu Ser145
150 155 160Pro Phe Leu Val Pro Met Ile
Ser Tyr Ala Ala Ser Ser Glu Thr Leu 165
170 175Ser Val Lys Arg Gln Tyr Pro Ser Phe Leu Arg Thr
Ile Pro Asn Asp 180 185 190Lys
Tyr Gln Val Glu Thr Met Val Leu Leu Leu Gln Lys Phe Gly Trp 195
200 205Thr Trp Ile Ser Leu Val Gly Ser Ser
Asp Asp Tyr Gly Gln Leu Gly 210 215
220Val Gln Ala Leu Glu Asn Gln Ala Thr Gly Gln Gly Ile Cys Ile Ala225
230 235 240Phe Lys Asp Ile
Met Pro Phe Ser Ala Gln Val Gly Asp Glu Arg Met 245
250 255Gln Cys Leu Met Arg His Leu Ala Gln Ala
Gly Ala Thr Val Val Val 260 265
270Val Phe Ser Ser Arg Gln Leu Ala Arg Val Phe Phe Glu Ser Val Val
275 280 285Leu Thr Asn Leu Thr Gly Lys
Val Trp Val Ala Ser Glu Ala Trp Ala 290 295
300Leu Ser Arg His Ile Thr Gly Val Pro Gly Ile Gln Arg Ile Gly
Met305 310 315 320Val Leu
Gly Val Ala Ile Gln Lys Arg Ala Val Pro Gly Leu Lys Ala
325 330 335Phe Glu Glu Ala Tyr Ala Arg
Ala Asp Lys Glu Ala Pro Arg Pro Cys 340 345
350His Lys Gly Ser Trp Cys Ser Ser Asn Gln Leu Cys Arg Glu
Cys Gln 355 360 365Ala Phe Met Ala
His Thr Met Pro Lys Leu Lys Ala Phe Ser Met Ser 370
375 380Ser Ala Tyr Asn Ala Tyr Arg Ala Val Tyr Ala Val
Ala His Gly Leu385 390 395
400His Gln Leu Leu Gly Cys Ala Ser Gly Ala Cys Ser Arg Gly Arg Val
405 410 415Tyr Pro Trp Gln Leu
Leu Glu Gln Ile His Lys Val His Phe Leu Leu 420
425 430His Lys Asp Thr Val Ala Phe Asn Asp Asn Arg Asp
Pro Leu Ser Ser 435 440 445Tyr Asn
Ile Ile Ala Trp Asp Trp Asn Gly Pro Lys Trp Thr Phe Thr 450
455 460Val Leu Gly Ser Ser Thr Trp Ser Pro Val Gln
Leu Asn Ile Asn Glu465 470 475
480Thr Lys Ile Gln Trp His Gly Lys Asp Asn Gln Val Pro Lys Ser Val
485 490 495Cys Ser Ser Asp
Cys Leu Glu Gly His Gln Arg Val Val Thr Gly Phe 500
505 510His His Cys Cys Phe Glu Cys Val Pro Cys Gly
Ala Gly Thr Phe Leu 515 520 525Asn
Lys Ser Asp Leu Tyr Arg Cys Gln Pro Cys Gly Lys Glu Glu Trp 530
535 540Ala Pro Glu Gly Ser Gln Thr Cys Phe Pro
Arg Thr Val Val Phe Leu545 550 555
560Ala Leu Arg Glu His Thr Ser Trp Val Leu Leu Ala Ala Asn Thr
Leu 565 570 575Leu Leu Leu
Leu Leu Leu Gly Thr Ala Gly Leu Phe Ala Trp His Leu 580
585 590Asp Thr Pro Val Val Arg Ser Ala Gly Gly
Arg Leu Cys Phe Leu Met 595 600
605Leu Gly Ser Leu Ala Ala Gly Ser Gly Ser Leu Tyr Gly Phe Phe Gly 610
615 620Glu Pro Thr Arg Pro Ala Cys Leu
Leu Arg Gln Ala Leu Phe Ala Leu625 630
635 640Gly Phe Thr Ile Phe Leu Ser Cys Leu Thr Val Arg
Ser Phe Gln Leu 645 650
655Ile Ile Ile Phe Lys Phe Ser Thr Lys Val Pro Thr Phe Tyr His Ala
660 665 670Trp Val Gln Asn His Gly
Ala Gly Leu Phe Val Met Ile Ser Ser Ala 675 680
685Ala Gln Leu Leu Ile Cys Leu Thr Trp Leu Val Val Trp Thr
Pro Leu 690 695 700Pro Ala Arg Glu Tyr
Gln Arg Phe Pro His Leu Val Met Leu Glu Cys705 710
715 720Thr Glu Thr Asn Ser Leu Gly Phe Ile Leu
Ala Phe Leu Tyr Asn Gly 725 730
735Leu Leu Ser Ile Ser Ala Phe Ala Cys Ser Tyr Leu Gly Lys Asp Leu
740 745 750Pro Glu Asn Tyr Asn
Glu Ala Lys Cys Val Thr Phe Ser Leu Leu Phe 755
760 765Asn Phe Val Ser Trp Ile Ala Phe Phe Thr Thr Ala
Ser Val Tyr Asp 770 775 780Gly Lys Tyr
Leu Pro Ala Ala Asn Met Met Ala Gly Leu Ser Ser Leu785
790 795 800Ser Ser Gly Phe Gly Gly Tyr
Phe Leu Pro Lys Cys Tyr Val Ile Leu 805
810 815Cys Arg Pro Asp Leu Asn Ser Thr Glu His Phe Gln
Ala Ser Ile Gln 820 825 830Asp
Tyr Thr Arg Arg Cys Gly Ser Thr 835
84013839PRTHomo sapiens 13Met Gly Pro Arg Ala Lys Thr Ile Ser Ser Leu Phe
Phe Leu Leu Trp1 5 10
15Val Leu Ala Glu Pro Ala Glu Asn Ser Asp Phe Tyr Leu Pro Gly Asp
20 25 30Tyr Leu Leu Gly Gly Leu Phe
Ser Leu His Ala Asn Met Lys Gly Ile 35 40
45Val His Leu Asn Phe Leu Gln Val Pro Met Cys Lys Glu Tyr Glu
Val 50 55 60Lys Val Ile Gly Tyr Asn
Leu Met Gln Ala Met Arg Phe Ala Val Glu65 70
75 80Glu Ile Asn Asn Asp Ser Ser Leu Leu Pro Gly
Val Leu Leu Gly Tyr 85 90
95Glu Ile Val Asp Val Cys Tyr Ile Ser Asn Asn Val Gln Pro Val Leu
100 105 110Tyr Phe Leu Ala His Glu
Asp Asn Leu Leu Pro Ile Gln Glu Asp Tyr 115 120
125Ser Asn Tyr Ile Ser Arg Val Val Ala Val Ile Gly Pro Asp
Asn Ser 130 135 140Glu Ser Val Met Thr
Val Ala Asn Phe Leu Ser Leu Phe Leu Leu Pro145 150
155 160Gln Ile Thr Tyr Ser Ala Ile Ser Asp Glu
Leu Arg Asp Lys Val Arg 165 170
175Phe Pro Ala Leu Leu Arg Thr Thr Pro Ser Ala Asp His His Ile Glu
180 185 190Ala Met Val Gln Leu
Met Leu His Phe Arg Trp Asn Trp Ile Ile Val 195
200 205Leu Val Ser Ser Asp Thr Tyr Gly Arg Asp Asn Gly
Gln Leu Leu Gly 210 215 220Glu Arg Val
Ala Arg Arg Asp Ile Cys Ile Ala Phe Gln Glu Thr Leu225
230 235 240Pro Thr Leu Gln Pro Asn Gln
Asn Met Thr Ser Glu Glu Arg Gln Arg 245
250 255Leu Val Thr Ile Val Asp Lys Leu Gln Gln Ser Thr
Ala Arg Val Val 260 265 270Val
Val Phe Ser Pro Asp Leu Thr Leu Tyr His Phe Phe Asn Glu Val 275
280 285Leu Arg Gln Asn Phe Thr Gly Ala Val
Trp Ile Ala Ser Glu Ser Trp 290 295
300Ala Ile Asp Pro Val Leu His Asn Leu Thr Glu Leu Arg His Leu Gly305
310 315 320Thr Phe Leu Gly
Ile Thr Ile Gln Ser Val Pro Ile Pro Gly Phe Ser 325
330 335Glu Phe Arg Glu Trp Gly Pro Gln Ala Gly
Pro Pro Pro Leu Ser Arg 340 345
350Thr Ser Gln Ser Tyr Thr Cys Asn Gln Glu Cys Asp Asn Cys Leu Asn
355 360 365Ala Thr Leu Ser Phe Asn Thr
Ile Leu Arg Leu Ser Gly Glu Arg Val 370 375
380Val Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala His Ala Leu
His385 390 395 400Ser Leu
Leu Gly Cys Asp Lys Ser Thr Cys Thr Lys Arg Val Val Tyr
405 410 415Pro Trp Gln Leu Leu Glu Glu
Ile Trp Lys Val Asn Phe Thr Leu Leu 420 425
430Asp His Gln Ile Phe Phe Asp Pro Gln Gly Asp Val Ala Leu
His Leu 435 440 445Glu Ile Val Gln
Trp Gln Trp Asp Arg Ser Gln Asn Pro Phe Gln Ser 450
455 460Val Ala Ser Tyr Tyr Pro Leu Gln Arg Gln Leu Lys
Asn Ile Gln Asp465 470 475
480Ile Ser Trp His Thr Ile Asn Asn Thr Ile Pro Met Ser Met Cys Ser
485 490 495Lys Arg Cys Gln Ser
Gly Gln Lys Lys Lys Pro Val Gly Ile His Val 500
505 510Cys Cys Phe Glu Cys Ile Asp Cys Leu Pro Gly Thr
Phe Leu Asn His 515 520 525Thr Glu
Asp Glu Tyr Glu Cys Gln Ala Cys Pro Asn Asn Glu Trp Ser 530
535 540Tyr Gln Ser Glu Thr Ser Cys Phe Lys Arg Gln
Leu Val Phe Leu Glu545 550 555
560Trp His Glu Ala Pro Thr Ile Ala Val Ala Leu Leu Ala Ala Leu Gly
565 570 575Phe Leu Ser Thr
Leu Ala Ile Leu Val Ile Phe Trp Arg His Phe Gln 580
585 590Thr Pro Ile Val Arg Ser Ala Gly Gly Pro Met
Cys Phe Leu Met Leu 595 600 605Thr
Leu Leu Leu Val Ala Tyr Met Val Val Pro Val Tyr Val Gly Pro 610
615 620Pro Lys Val Ser Thr Cys Leu Cys Arg Gln
Ala Leu Phe Pro Leu Cys625 630 635
640Phe Thr Ile Cys Ile Ser Cys Ile Ala Val Arg Ser Phe Gln Ile
Val 645 650 655Cys Ala Phe
Lys Met Ala Ser Arg Phe Pro Arg Ala Tyr Ser Tyr Trp 660
665 670Val Arg Tyr Gln Gly Pro Tyr Val Ser Met
Ala Phe Ile Thr Val Leu 675 680
685Lys Met Val Ile Val Val Ile Gly Met Leu Ala Thr Gly Leu Ser Pro 690
695 700Thr Thr Arg Thr Asp Pro Asp Asp
Pro Lys Ile Thr Ile Val Ser Cys705 710
715 720Asn Pro Asn Tyr Arg Asn Ser Leu Leu Phe Asn Thr
Ser Leu Asp Leu 725 730
735Leu Leu Ser Val Val Gly Phe Ser Phe Ala Tyr Met Gly Lys Glu Leu
740 745 750Pro Thr Asn Tyr Asn Glu
Ala Lys Phe Ile Thr Leu Ser Met Thr Phe 755 760
765Tyr Phe Thr Ser Ser Val Ser Leu Cys Thr Phe Met Ser Ala
Tyr Ser 770 775 780Gly Val Leu Val Thr
Ile Val Asp Leu Leu Val Thr Val Leu Asn Leu785 790
795 800Leu Ala Ile Ser Leu Gly Tyr Phe Gly Pro
Lys Cys Tyr Met Ile Leu 805 810
815Phe Tyr Pro Glu Arg Asn Thr Pro Ala Tyr Phe Asn Ser Met Ile Gln
820 825 830Gly Tyr Thr Met Arg
Arg Asp 83514839PRTHomo sapiens 14Met Gly Pro Arg Ala Lys Thr Ile
Cys Ser Leu Phe Phe Leu Leu Trp1 5 10
15Val Leu Ala Glu Pro Ala Glu Asn Ser Asp Phe Tyr Leu Pro
Gly Asp 20 25 30Tyr Leu Leu
Gly Gly Leu Phe Ser Leu His Ala Asn Met Lys Gly Ile 35
40 45Val His Leu Asn Phe Leu Gln Val Pro Met Cys
Lys Glu Tyr Glu Val 50 55 60Lys Val
Ile Gly Tyr Asn Leu Met Gln Ala Met Arg Phe Ala Val Glu65
70 75 80Glu Ile Asn Asn Asp Ser Ser
Leu Leu Pro Gly Val Leu Leu Gly Tyr 85 90
95Glu Ile Val Asp Val Cys Tyr Ile Ser Asn Asn Val Gln
Pro Val Leu 100 105 110Tyr Phe
Leu Ala His Glu Asp Asn Leu Leu Pro Ile Gln Glu Asp Tyr 115
120 125Ser Asn Tyr Ile Ser Arg Val Val Ala Val
Ile Gly Pro Asp Asn Ser 130 135 140Glu
Ser Val Met Thr Val Ala Asn Phe Leu Ser Leu Phe Leu Leu Pro145
150 155 160Gln Ile Thr Tyr Ser Ala
Ile Ser Asp Glu Leu Arg Asp Lys Val Arg 165
170 175Phe Pro Ala Leu Leu Arg Thr Thr Pro Ser Ala Asp
His His Val Glu 180 185 190Ala
Met Val Gln Leu Met Leu His Phe Arg Trp Asn Trp Ile Ile Val 195
200 205Leu Val Ser Ser Asp Thr Tyr Gly Arg
Asp Asn Gly Gln Leu Leu Gly 210 215
220Glu Arg Val Ala Arg Arg Asp Ile Cys Ile Ala Phe Gln Glu Thr Leu225
230 235 240Pro Thr Leu Gln
Pro Asn Gln Asn Met Thr Ser Glu Glu Arg Gln Arg 245
250 255Leu Val Thr Ile Val Asp Lys Leu Gln Gln
Ser Thr Ala Arg Val Val 260 265
270Val Val Phe Ser Pro Asp Leu Thr Leu Tyr His Phe Phe Asn Glu Val
275 280 285Leu Arg Gln Asn Phe Thr Gly
Ala Val Trp Ile Ala Ser Glu Ser Trp 290 295
300Ala Ile Asp Pro Val Leu His Asn Leu Thr Glu Leu Gly His Leu
Gly305 310 315 320Thr Phe
Leu Gly Ile Thr Ile Gln Ser Val Pro Ile Pro Gly Phe Ser
325 330 335Glu Phe Arg Glu Trp Gly Pro
Gln Ala Gly Pro Pro Pro Leu Ser Arg 340 345
350Thr Ser Gln Ser Tyr Thr Cys Asn Gln Glu Cys Asp Asn Cys
Leu Asn 355 360 365Ala Thr Leu Ser
Phe Asn Thr Ile Leu Arg Leu Ser Gly Glu Arg Val 370
375 380Val Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala
His Ala Leu His385 390 395
400Ser Leu Leu Gly Cys Asp Lys Ser Thr Cys Thr Lys Arg Val Val Tyr
405 410 415Pro Trp Gln Leu Leu
Glu Glu Ile Trp Lys Val Asn Phe Thr Leu Leu 420
425 430Asp His Gln Ile Phe Phe Asp Pro Gln Gly Asp Val
Ala Leu His Leu 435 440 445Glu Ile
Val Gln Trp Gln Trp Asp Arg Ser Gln Asn Pro Phe Gln Ser 450
455 460Val Ala Ser Tyr Tyr Pro Leu Gln Arg Gln Leu
Lys Asn Ile Gln Asp465 470 475
480Ile Ser Trp His Thr Val Asn Asn Thr Ile Pro Met Ser Met Cys Ser
485 490 495Lys Arg Cys Gln
Ser Gly Gln Lys Lys Lys Pro Val Gly Ile His Val 500
505 510Cys Cys Phe Glu Cys Ile Asp Cys Leu Pro Gly
Thr Phe Leu Asn His 515 520 525Thr
Glu Asp Glu Tyr Glu Cys Gln Ala Cys Pro Asn Asn Glu Trp Ser 530
535 540Tyr Gln Ser Glu Thr Ser Cys Phe Lys Arg
Gln Leu Val Phe Leu Glu545 550 555
560Trp His Glu Ala Pro Thr Ile Ala Val Ala Leu Leu Ala Ala Leu
Gly 565 570 575Phe Leu Ser
Thr Leu Ala Ile Leu Val Ile Phe Trp Arg His Phe Gln 580
585 590Thr Pro Ile Val Arg Ser Ala Gly Gly Pro
Met Cys Phe Leu Met Leu 595 600
605Thr Leu Leu Leu Val Ala Tyr Met Val Val Pro Val Tyr Val Gly Pro 610
615 620Pro Lys Val Ser Thr Cys Leu Cys
Arg Gln Ala Leu Phe Pro Leu Cys625 630
635 640Phe Thr Ile Cys Ile Ser Cys Ile Ala Val Arg Ser
Phe Gln Ile Val 645 650
655Cys Ala Phe Lys Met Ala Ser Arg Phe Pro Arg Ala Tyr Ser Tyr Trp
660 665 670Val Arg Tyr Gln Gly Pro
Tyr Val Ser Met Ala Phe Ile Thr Val Leu 675 680
685Lys Met Val Ile Val Val Ile Gly Met Leu Ala Thr Gly Leu
Ser Pro 690 695 700Thr Thr Arg Thr Asp
Pro Asp Asp Pro Lys Ile Thr Ile Val Ser Cys705 710
715 720Asn Pro Asn Tyr Arg Asn Ser Leu Leu Phe
Asn Thr Ser Leu Asp Leu 725 730
735Leu Leu Ser Val Val Gly Phe Ser Phe Ala Tyr Met Gly Lys Glu Leu
740 745 750Pro Thr Asn Tyr Asn
Glu Ala Lys Phe Ile Thr Leu Ser Met Thr Phe 755
760 765Tyr Phe Thr Ser Ser Val Ser Leu Cys Thr Phe Met
Ser Ala Tyr Ser 770 775 780Gly Val Leu
Val Thr Ile Val Asp Leu Leu Val Thr Val Leu Asn Leu785
790 795 800Leu Ala Ile Ser Leu Gly Tyr
Phe Gly Pro Lys Cys Tyr Met Ile Leu 805
810 815Phe Tyr Pro Glu Arg Asn Thr Pro Ala Tyr Phe Asn
Ser Met Ile Gln 820 825 830Gly
Tyr Thr Met Arg Arg Asp 83515843PRTMus musculus 15Met Gly Pro Gln
Ala Arg Thr Leu His Leu Leu Phe Leu Leu Leu His1 5
10 15Ala Leu Pro Lys Pro Val Met Leu Val Gly
Asn Ser Asp Phe His Leu 20 25
30Ala Gly Asp Tyr Leu Leu Gly Gly Leu Phe Thr Leu His Ala Asn Val
35 40 45Lys Ser Val Ser His Leu Ser Tyr
Leu Gln Val Pro Lys Cys Asn Glu 50 55
60Tyr Asn Met Lys Val Leu Gly Tyr Asn Leu Met Gln Ala Met Arg Phe65
70 75 80Ala Val Glu Glu Ile
Asn Asn Cys Ser Ser Leu Leu Pro Gly Val Leu 85
90 95Leu Gly Tyr Glu Met Val Asp Val Cys Tyr Leu
Ser Asn Asn Ile Gln 100 105
110Pro Gly Leu Tyr Phe Leu Ser Gln Ile Asp Asp Phe Leu Pro Ile Leu
115 120 125Lys Asp Tyr Ser Gln Tyr Arg
Pro Gln Val Val Ala Val Ile Gly Pro 130 135
140Asp Asn Ser Glu Ser Ala Ile Thr Val Ser Asn Ile Leu Ser Tyr
Phe145 150 155 160Leu Val
Pro Gln Val Thr Tyr Ser Ala Ile Thr Asp Lys Leu Arg Asp
165 170 175Lys Arg Arg Phe Pro Ala Met
Leu Arg Thr Val Pro Ser Ala Thr His 180 185
190His Ile Glu Ala Met Val Gln Leu Met Val His Phe Gln Trp
Asn Trp 195 200 205Ile Val Val Leu
Val Ser Asp Asp Asp Tyr Gly Arg Glu Asn Ser His 210
215 220Leu Leu Ser Gln Arg Leu Thr Asn Thr Gly Asp Ile
Cys Ile Ala Phe225 230 235
240Gln Glu Val Leu Pro Val Pro Glu Pro Asn Gln Ala Val Arg Pro Glu
245 250 255Glu Gln Asp Gln Leu
Asp Asn Ile Leu Asp Lys Leu Arg Arg Thr Ser 260
265 270Ala Arg Val Val Val Ile Phe Ser Pro Glu Leu Ser
Leu His Asn Phe 275 280 285Phe Arg
Glu Val Leu Arg Trp Asn Phe Thr Gly Phe Val Trp Ile Ala 290
295 300Ser Glu Ser Trp Ala Ile Asp Pro Val Leu His
Asn Leu Thr Glu Leu305 310 315
320Arg His Thr Gly Thr Phe Leu Gly Val Thr Ile Gln Arg Val Ser Ile
325 330 335Pro Gly Phe Ser
Gln Phe Arg Val Arg His Asp Lys Pro Glu Tyr Pro 340
345 350Met Pro Asn Glu Thr Ser Leu Arg Thr Thr Cys
Asn Gln Asp Cys Asp 355 360 365Ala
Cys Met Asn Ile Thr Glu Ser Phe Asn Asn Val Leu Met Leu Ser 370
375 380Gly Glu Arg Val Val Tyr Ser Val Tyr Ser
Ala Val Tyr Ala Val Ala385 390 395
400His Thr Leu His Arg Leu Leu His Cys Asn Gln Val Arg Cys Thr
Lys 405 410 415Gln Ile Val
Tyr Pro Trp Gln Leu Leu Arg Glu Ile Trp His Val Asn 420
425 430Phe Thr Leu Leu Gly Asn Gln Leu Phe Phe
Asp Glu Gln Gly Asp Met 435 440
445Pro Met Leu Leu Asp Ile Ile Gln Trp Gln Trp Gly Leu Ser Gln Asn 450
455 460Pro Phe Gln Ser Ile Ala Ser Tyr
Ser Pro Thr Glu Thr Arg Leu Thr465 470
475 480Tyr Ile Ser Asn Val Ser Trp Tyr Thr Pro Asn Asn
Thr Val Pro Ile 485 490
495Ser Met Cys Ser Lys Ser Cys Gln Pro Gly Gln Met Lys Lys Pro Ile
500 505 510Gly Leu His Pro Cys Cys
Phe Glu Cys Val Asp Cys Pro Pro Gly Thr 515 520
525Tyr Leu Asn Arg Ser Val Asp Glu Phe Asn Cys Leu Ser Cys
Pro Gly 530 535 540Ser Met Trp Ser Tyr
Lys Asn Asn Ile Ala Cys Phe Lys Arg Arg Leu545 550
555 560Ala Phe Leu Glu Trp His Glu Val Pro Thr
Ile Val Val Thr Ile Leu 565 570
575Ala Ala Leu Gly Phe Ile Ser Thr Leu Ala Ile Leu Leu Ile Phe Trp
580 585 590Arg His Phe Gln Thr
Pro Met Val Arg Ser Ala Gly Gly Pro Met Cys 595
600 605Phe Leu Met Leu Val Pro Leu Leu Leu Ala Phe Gly
Met Val Pro Val 610 615 620Tyr Val Gly
Pro Pro Thr Val Phe Ser Cys Phe Cys Arg Gln Ala Phe625
630 635 640Phe Thr Val Cys Phe Ser Val
Cys Leu Ser Cys Ile Thr Val Arg Ser 645
650 655Phe Gln Ile Val Cys Val Phe Lys Met Ala Arg Arg
Leu Pro Ser Ala 660 665 670Tyr
Gly Phe Trp Met Arg Tyr His Gly Pro Tyr Val Phe Val Ala Phe 675
680 685Ile Thr Ala Val Lys Val Ala Leu Val
Ala Gly Asn Met Leu Ala Thr 690 695
700Thr Ile Asn Pro Ile Gly Arg Thr Asp Pro Asp Asp Pro Asn Ile Ile705
710 715 720Ile Leu Ser Cys
His Pro Asn Tyr Arg Asn Gly Leu Leu Phe Asn Thr 725
730 735Ser Met Asp Leu Leu Leu Ser Val Leu Gly
Phe Ser Phe Ala Tyr Val 740 745
750Gly Lys Glu Leu Pro Thr Asn Tyr Asn Glu Ala Lys Phe Ile Thr Leu
755 760 765Ser Met Thr Phe Ser Phe Thr
Ser Ser Ile Ser Leu Cys Thr Phe Met 770 775
780Ser Val His Asp Gly Val Leu Val Thr Ile Met Asp Leu Leu Val
Thr785 790 795 800Val Leu
Asn Phe Leu Ala Ile Gly Leu Gly Tyr Phe Gly Pro Lys Cys
805 810 815Tyr Met Ile Leu Phe Tyr Pro
Glu Arg Asn Thr Ser Ala Tyr Phe Asn 820 825
830Ser Met Ile Gln Gly Tyr Thr Met Arg Lys Ser 835
84016843PRTMus musculus 16Met Gly Pro Gln Ala Arg Thr Leu
His Leu Leu Phe Leu Leu Leu His1 5 10
15Ala Leu Pro Lys Pro Val Met Leu Val Gly Asn Ser Asp Phe
His Leu 20 25 30Ala Gly Asp
Tyr Leu Leu Gly Gly Leu Phe Thr Leu His Ala Asn Val 35
40 45Lys Ser Val Ser His Leu Ser Tyr Leu Gln Val
Pro Lys Cys Asn Glu 50 55 60Tyr Asn
Met Lys Val Leu Gly Tyr Asn Leu Met Gln Ala Met Arg Phe65
70 75 80Ala Val Glu Glu Ile Asn Asn
Cys Ser Ser Leu Leu Pro Gly Val Leu 85 90
95Leu Gly Tyr Glu Met Val Asp Val Cys Tyr Leu Ser Asn
Asn Ile Gln 100 105 110Pro Gly
Leu Tyr Phe Leu Ser Gln Ile Asp Asp Phe Leu Pro Ile Leu 115
120 125Lys Asp Tyr Ser Gln Tyr Arg Pro Gln Val
Val Ala Val Ile Gly Pro 130 135 140Asp
Asn Ser Glu Ser Ala Ile Thr Val Ser Asn Ile Leu Ser Tyr Phe145
150 155 160Leu Val Pro Gln Val Thr
Tyr Ser Ala Ile Thr Asp Lys Leu Arg Asp 165
170 175Lys Arg Arg Phe Pro Ala Met Leu Arg Thr Val Pro
Ser Ala Thr His 180 185 190His
Ile Glu Ala Met Val Gln Leu Met Val His Phe Gln Trp Asn Trp 195
200 205Ile Val Val Leu Val Ser Asp Asp Asp
Tyr Gly Arg Glu Asn Ser His 210 215
220Leu Leu Ser Gln Arg Leu Thr Asn Thr Gly Asp Ile Cys Ile Ala Phe225
230 235 240Gln Glu Val Leu
Pro Val Pro Glu Pro Asn Gln Ala Val Arg Pro Glu 245
250 255Glu Gln Asp Gln Leu Asp Asn Ile Leu Asp
Lys Leu Arg Arg Thr Ser 260 265
270Ala Arg Val Val Val Ile Phe Ser Pro Glu Leu Ser Leu His Asn Phe
275 280 285Phe Arg Glu Val Leu Arg Trp
Asn Phe Thr Gly Phe Val Trp Ile Ala 290 295
300Ser Glu Ser Trp Ala Ile Asp Pro Val Leu His Asn Leu Thr Glu
Leu305 310 315 320Arg His
Thr Gly Thr Phe Leu Gly Val Thr Ile Gln Arg Val Ser Ile
325 330 335Pro Gly Phe Ser Gln Phe Arg
Val Arg His Asp Lys Pro Glu Tyr Pro 340 345
350Met Pro Asn Glu Thr Ser Leu Arg Thr Thr Cys Asn Gln Asp
Cys Asp 355 360 365Ala Cys Met Asn
Ile Thr Glu Ser Phe Asn Asn Val Leu Met Leu Ser 370
375 380Gly Glu Arg Val Val Tyr Ser Val Tyr Ser Ala Val
Tyr Ala Val Ala385 390 395
400His Thr Leu His Arg Leu Leu His Cys Asn Gln Val Arg Cys Thr Lys
405 410 415Gln Ile Val Tyr Pro
Trp Gln Leu Leu Arg Glu Ile Trp His Val Asn 420
425 430Phe Thr Leu Leu Gly Asn Gln Leu Phe Phe Asp Glu
Gln Gly Asp Met 435 440 445Pro Met
Leu Leu Asp Ile Ile Gln Trp Gln Trp Gly Leu Ser Gln Asn 450
455 460Pro Phe Gln Ser Ile Ala Ser Tyr Ser Pro Thr
Glu Thr Arg Leu Thr465 470 475
480Tyr Ile Ser Asn Val Ser Trp Tyr Thr Pro Asn Asn Thr Val Pro Ile
485 490 495Ser Met Cys Ser
Lys Ser Cys Gln Pro Gly Gln Met Lys Lys Pro Ile 500
505 510Gly Leu His Pro Cys Cys Phe Glu Cys Val Asp
Cys Pro Pro Gly Thr 515 520 525Tyr
Leu Asn Arg Ser Val Asp Glu Phe Asn Cys Leu Ser Cys Pro Gly 530
535 540Ser Met Trp Ser Tyr Lys Asn Asn Ile Ala
Cys Phe Lys Arg Arg Leu545 550 555
560Ala Phe Leu Glu Trp His Glu Val Pro Thr Ile Val Val Thr Ile
Leu 565 570 575Ala Ala Leu
Gly Phe Ile Ser Thr Leu Ala Ile Leu Leu Ile Phe Trp 580
585 590Arg His Phe Gln Thr Pro Met Val Arg Ser
Ala Gly Gly Pro Met Cys 595 600
605Phe Leu Met Leu Val Pro Leu Leu Leu Ala Phe Gly Met Val Pro Val 610
615 620Tyr Val Gly Pro Pro Thr Val Phe
Ser Cys Phe Cys Arg Gln Ala Phe625 630
635 640Phe Thr Val Cys Phe Ser Val Cys Leu Ser Cys Ile
Thr Val Arg Ser 645 650
655Phe Gln Ile Val Cys Val Phe Lys Met Ala Arg Arg Leu Pro Ser Ala
660 665 670Tyr Gly Phe Trp Met Arg
Tyr His Gly Pro Tyr Val Phe Val Ala Phe 675 680
685Ile Thr Ala Val Lys Val Ala Leu Val Ala Gly Asn Met Leu
Ala Thr 690 695 700Thr Ile Asn Pro Ile
Gly Arg Thr Asp Pro Asp Asp Pro Asn Ile Ile705 710
715 720Ile Leu Ser Cys His Pro Asn Tyr Arg Asn
Gly Leu Leu Phe Asn Thr 725 730
735Ser Met Asp Leu Leu Leu Ser Val Leu Gly Phe Ser Phe Ala Tyr Val
740 745 750Gly Lys Glu Leu Pro
Thr Asn Tyr Asn Glu Ala Lys Phe Ile Thr Leu 755
760 765Ser Met Thr Phe Ser Phe Thr Ser Ser Ile Ser Leu
Cys Thr Phe Met 770 775 780Ser Val His
Asp Gly Val Leu Val Thr Ile Met Asp Leu Leu Val Thr785
790 795 800Val Leu Asn Phe Leu Ala Ile
Gly Leu Gly Tyr Phe Gly Pro Lys Cys 805
810 815Tyr Met Ile Leu Phe Tyr Pro Glu Arg Asn Thr Ser
Ala Tyr Phe Asn 820 825 830Ser
Met Ile Gln Gly Tyr Thr Met Arg Lys Ser 835
84017840PRTRattus norvegicus 17Met Leu Phe Trp Ala Ala His Leu Leu Leu
Ser Leu Gln Leu Val Tyr1 5 10
15Cys Trp Ala Phe Ser Cys Gln Arg Thr Glu Ser Ser Pro Gly Phe Ser
20 25 30Leu Pro Gly Asp Phe Leu
Leu Ala Gly Leu Phe Ser Leu His Gly Asp 35 40
45Cys Leu Gln Val Arg His Arg Pro Leu Val Thr Ser Cys Asp
Arg Pro 50 55 60Asp Ser Phe Asn Gly
His Gly Tyr His Leu Phe Gln Ala Met Arg Phe65 70
75 80Thr Val Glu Glu Ile Asn Asn Ser Ser Ala
Leu Leu Pro Asn Ile Thr 85 90
95Leu Gly Tyr Glu Leu Tyr Asp Val Cys Ser Glu Ser Ala Asn Val Tyr
100 105 110Ala Thr Leu Arg Val
Leu Ala Leu Gln Gly Pro Arg His Ile Glu Ile 115
120 125Gln Lys Asp Leu Arg Asn His Ser Ser Lys Val Val
Ala Phe Ile Gly 130 135 140Pro Asp Asn
Thr Asp His Ala Val Thr Thr Ala Ala Leu Leu Gly Pro145
150 155 160Phe Leu Met Pro Leu Val Ser
Tyr Glu Ala Ser Ser Val Val Leu Ser 165
170 175Ala Lys Arg Lys Phe Pro Ser Phe Leu Arg Thr Val
Pro Ser Asp Arg 180 185 190His
Gln Val Glu Val Met Val Gln Leu Leu Gln Ser Phe Gly Trp Val 195
200 205Trp Ile Ser Leu Ile Gly Ser Tyr Gly
Asp Tyr Gly Gln Leu Gly Val 210 215
220Gln Ala Leu Glu Glu Leu Ala Val Pro Arg Gly Ile Cys Val Ala Phe225
230 235 240Lys Asp Ile Val
Pro Phe Ser Ala Arg Val Gly Asp Pro Arg Met Gln 245
250 255Ser Met Met Gln His Leu Ala Gln Ala Arg
Thr Thr Val Val Val Val 260 265
270Phe Ser Asn Arg His Leu Ala Arg Val Phe Phe Arg Ser Val Val Leu
275 280 285Ala Asn Leu Thr Gly Lys Val
Trp Val Ala Ser Glu Asp Trp Ala Ile 290 295
300Ser Thr Tyr Ile Thr Ser Val Thr Gly Ile Gln Gly Ile Gly Thr
Val305 310 315 320Leu Gly
Val Ala Val Gln Gln Arg Gln Val Pro Gly Leu Lys Glu Phe
325 330 335Glu Glu Ser Tyr Val Arg Ala
Val Thr Ala Ala Pro Ser Ala Cys Pro 340 345
350Glu Gly Ser Trp Cys Ser Thr Asn Gln Leu Cys Arg Glu Cys
His Thr 355 360 365Phe Thr Thr Arg
Asn Met Pro Thr Leu Gly Ala Phe Ser Met Ser Ala 370
375 380Ala Tyr Arg Val Tyr Glu Ala Val Tyr Ala Val Ala
His Gly Leu His385 390 395
400Gln Leu Leu Gly Cys Thr Ser Glu Ile Cys Ser Arg Gly Pro Val Tyr
405 410 415Pro Trp Gln Leu Leu
Gln Gln Ile Tyr Lys Val Asn Phe Leu Leu His 420
425 430Glu Asn Thr Val Ala Phe Asp Asp Asn Gly Asp Thr
Leu Gly Tyr Tyr 435 440 445Asp Ile
Ile Ala Trp Asp Trp Asn Gly Pro Glu Trp Thr Phe Glu Ile 450
455 460Ile Gly Ser Ala Ser Leu Ser Pro Val His Leu
Asp Ile Asn Lys Thr465 470 475
480Lys Ile Gln Trp His Gly Lys Asn Asn Gln Val Pro Val Ser Val Cys
485 490 495Thr Thr Asp Cys
Leu Ala Gly His His Arg Val Val Val Gly Ser His 500
505 510His Cys Cys Phe Glu Cys Val Pro Cys Glu Ala
Gly Thr Phe Leu Asn 515 520 525Met
Ser Glu Leu His Ile Cys Gln Pro Cys Gly Thr Glu Glu Trp Ala 530
535 540Pro Lys Glu Ser Thr Thr Cys Phe Pro Arg
Thr Val Glu Phe Leu Ala545 550 555
560Trp His Glu Pro Ile Ser Leu Val Leu Ile Ala Ala Asn Thr Leu
Leu 565 570 575Leu Leu Leu
Leu Val Gly Thr Ala Gly Leu Phe Ala Trp His Phe His 580
585 590Thr Pro Val Val Arg Ser Ala Gly Gly Arg
Leu Cys Phe Leu Met Leu 595 600
605Gly Ser Leu Val Ala Gly Ser Cys Ser Phe Tyr Ser Phe Phe Gly Glu 610
615 620Pro Thr Val Pro Ala Cys Leu Leu
Arg Gln Pro Leu Phe Ser Leu Gly625 630
635 640Phe Ala Ile Phe Leu Ser Cys Leu Thr Ile Arg Ser
Phe Gln Leu Val 645 650
655Ile Ile Phe Lys Phe Ser Thr Lys Val Pro Thr Phe Tyr Arg Thr Trp
660 665 670Ala Gln Asn His Gly Ala
Gly Leu Phe Val Ile Val Ser Ser Thr Val 675 680
685His Leu Leu Ile Cys Leu Thr Trp Leu Val Met Trp Thr Pro
Arg Pro 690 695 700Thr Arg Glu Tyr Gln
Arg Phe Pro His Leu Val Ile Leu Glu Cys Thr705 710
715 720Glu Val Asn Ser Val Gly Phe Leu Leu Ala
Phe Thr His Asn Ile Leu 725 730
735Leu Ser Ile Ser Thr Phe Val Cys Ser Tyr Leu Gly Lys Glu Leu Pro
740 745 750Glu Asn Tyr Asn Glu
Ala Lys Cys Val Thr Phe Ser Leu Leu Leu Asn 755
760 765Phe Val Ser Trp Ile Ala Phe Phe Thr Met Ala Ser
Ile Tyr Gln Gly 770 775 780Ser Tyr Leu
Pro Ala Val Asn Val Leu Ala Gly Leu Thr Thr Leu Ser785
790 795 800Gly Gly Phe Ser Gly Tyr Phe
Leu Pro Lys Cys Tyr Val Ile Leu Cys 805
810 815Arg Pro Glu Leu Asn Asn Thr Glu His Phe Gln Ala
Ser Ile Gln Asp 820 825 830Tyr
Thr Arg Arg Cys Gly Thr Thr 835 84018843PRTRattus
norvegicus 18Met Gly Pro Gln Ala Arg Thr Leu Cys Leu Leu Ser Leu Leu Leu
His1 5 10 15Val Leu Pro
Lys Pro Gly Lys Leu Val Glu Asn Ser Asp Phe His Leu 20
25 30Ala Gly Asp Tyr Leu Leu Gly Gly Leu Phe
Thr Leu His Ala Asn Val 35 40
45Lys Ser Ile Ser His Leu Ser Tyr Leu Gln Val Pro Lys Cys Asn Glu 50
55 60Phe Thr Met Lys Val Leu Gly Tyr Asn
Leu Met Gln Ala Met Arg Phe65 70 75
80Ala Val Glu Glu Ile Asn Asn Cys Ser Ser Leu Leu Pro Gly
Val Leu 85 90 95Leu Gly
Tyr Glu Met Val Asp Val Cys Tyr Leu Ser Asn Asn Ile His 100
105 110Pro Gly Leu Tyr Phe Leu Ala Gln Asp
Asp Asp Leu Leu Pro Ile Leu 115 120
125Lys Asp Tyr Ser Gln Tyr Met Pro His Val Val Ala Val Ile Gly Pro
130 135 140Asp Asn Ser Glu Ser Ala Ile
Thr Val Ser Asn Ile Leu Ser His Phe145 150
155 160Leu Ile Pro Gln Ile Thr Tyr Ser Ala Ile Ser Asp
Lys Leu Arg Asp 165 170
175Lys Arg His Phe Pro Ser Met Leu Arg Thr Val Pro Ser Ala Thr His
180 185 190His Ile Glu Ala Met Val
Gln Leu Met Val His Phe Gln Trp Asn Trp 195 200
205Ile Val Val Leu Val Ser Asp Asp Asp Tyr Gly Arg Glu Asn
Ser His 210 215 220Leu Leu Ser Gln Arg
Leu Thr Lys Thr Ser Asp Ile Cys Ile Ala Phe225 230
235 240Gln Glu Val Leu Pro Ile Pro Glu Ser Ser
Gln Val Met Arg Ser Glu 245 250
255Glu Gln Arg Gln Leu Asp Asn Ile Leu Asp Lys Leu Arg Arg Thr Ser
260 265 270Ala Arg Val Val Val
Val Phe Ser Pro Glu Leu Ser Leu Tyr Ser Phe 275
280 285Phe His Glu Val Leu Arg Trp Asn Phe Thr Gly Phe
Val Trp Ile Ala 290 295 300Ser Glu Ser
Trp Ala Ile Asp Pro Val Leu His Asn Leu Thr Glu Leu305
310 315 320Arg His Thr Gly Thr Phe Leu
Gly Val Thr Ile Gln Arg Val Ser Ile 325
330 335Pro Gly Phe Ser Gln Phe Arg Val Arg Arg Asp Lys
Pro Gly Tyr Pro 340 345 350Val
Pro Asn Thr Thr Asn Leu Arg Thr Thr Cys Asn Gln Asp Cys Asp 355
360 365Ala Cys Leu Asn Thr Thr Lys Ser Phe
Asn Asn Ile Leu Ile Leu Ser 370 375
380Gly Glu Arg Val Val Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala385
390 395 400His Ala Leu His
Arg Leu Leu Gly Cys Asn Arg Val Arg Cys Thr Lys 405
410 415Gln Lys Val Tyr Pro Trp Gln Leu Leu Arg
Glu Ile Trp His Val Asn 420 425
430Phe Thr Leu Leu Gly Asn Arg Leu Phe Phe Asp Gln Gln Gly Asp Met
435 440 445Pro Met Leu Leu Asp Ile Ile
Gln Trp Gln Trp Asp Leu Ser Gln Asn 450 455
460Pro Phe Gln Ser Ile Ala Ser Tyr Ser Pro Thr Ser Lys Arg Leu
Thr465 470 475 480Tyr Ile
Asn Asn Val Ser Trp Tyr Thr Pro Asn Asn Thr Val Pro Val
485 490 495Ser Met Cys Ser Lys Ser Cys
Gln Pro Gly Gln Met Lys Lys Ser Val 500 505
510Gly Leu His Pro Cys Cys Phe Glu Cys Leu Asp Cys Met Pro
Gly Thr 515 520 525Tyr Leu Asn Arg
Ser Ala Asp Glu Phe Asn Cys Leu Ser Cys Pro Gly 530
535 540Ser Met Trp Ser Tyr Lys Asn Asp Ile Thr Cys Phe
Gln Arg Arg Pro545 550 555
560Thr Phe Leu Glu Trp His Glu Val Pro Thr Ile Val Val Ala Ile Leu
565 570 575Ala Ala Leu Gly Phe
Phe Ser Thr Leu Ala Ile Leu Phe Ile Phe Trp 580
585 590Arg His Phe Gln Thr Pro Met Val Arg Ser Ala Gly
Gly Pro Met Cys 595 600 605Phe Leu
Met Leu Val Pro Leu Leu Leu Ala Phe Gly Met Val Pro Val 610
615 620Tyr Val Gly Pro Pro Thr Val Phe Ser Cys Phe
Cys Arg Gln Ala Phe625 630 635
640Phe Thr Val Cys Phe Ser Ile Cys Leu Ser Cys Ile Thr Val Arg Ser
645 650 655Phe Gln Ile Val
Cys Val Phe Lys Met Ala Arg Arg Leu Pro Ser Ala 660
665 670Tyr Ser Phe Trp Met Arg Tyr His Gly Pro Tyr
Val Phe Val Ala Phe 675 680 685Ile
Thr Ala Ile Lys Val Ala Leu Val Val Gly Asn Met Leu Ala Thr 690
695 700Thr Ile Asn Pro Ile Gly Arg Thr Asp Pro
Asp Asp Pro Asn Ile Met705 710 715
720Ile Leu Ser Cys His Pro Asn Tyr Arg Asn Gly Leu Leu Phe Asn
Thr 725 730 735Ser Met Asp
Leu Leu Leu Ser Val Leu Gly Phe Ser Phe Ala Tyr Met 740
745 750Gly Lys Glu Leu Pro Thr Asn Tyr Asn Glu
Ala Lys Phe Ile Thr Leu 755 760
765Ser Met Thr Phe Ser Phe Thr Ser Ser Ile Ser Leu Cys Thr Phe Met 770
775 780Ser Val His Asp Gly Val Leu Val
Thr Ile Met Asp Leu Leu Val Thr785 790
795 800Val Leu Asn Phe Leu Ala Ile Gly Leu Gly Tyr Phe
Gly Pro Lys Cys 805 810
815Tyr Met Ile Leu Phe Tyr Pro Glu Arg Asn Thr Ser Ala Tyr Phe Asn
820 825 830Ser Met Ile Gln Gly Tyr
Thr Met Arg Lys Ser 835 84019842PRTMus musculus
19Met Leu Phe Trp Ala Ala His Leu Leu Leu Ser Leu Gln Leu Ala Val1
5 10 15Ala Tyr Cys Trp Ala Phe
Ser Cys Gln Arg Thr Glu Ser Ser Pro Gly 20 25
30Phe Ser Leu Pro Gly Asp Phe Leu Leu Ala Gly Leu Phe
Ser Leu His 35 40 45Ala Asp Cys
Leu Gln Val Arg His Arg Pro Leu Val Thr Ser Cys Asp 50
55 60Arg Ser Asp Ser Phe Asn Gly His Gly Tyr His Leu
Phe Gln Ala Met65 70 75
80Arg Phe Thr Val Glu Glu Ile Asn Asn Ser Thr Ala Leu Leu Pro Asn
85 90 95Ile Thr Leu Gly Tyr Glu
Leu Tyr Asp Val Cys Ser Glu Ser Ser Asn 100
105 110Val Tyr Ala Thr Leu Arg Val Leu Ala Gln Gln Gly
Thr Gly His Leu 115 120 125Glu Met
Gln Arg Asp Leu Arg Asn His Ser Ser Lys Val Val Ala Leu 130
135 140Ile Gly Pro Asp Asn Thr Asp His Ala Val Thr
Thr Ala Ala Leu Leu145 150 155
160Ser Pro Phe Leu Met Pro Leu Val Ser Tyr Glu Ala Ser Ser Val Ile
165 170 175Leu Ser Gly Lys
Arg Lys Phe Pro Ser Phe Leu Arg Thr Ile Pro Ser 180
185 190Asp Lys Tyr Gln Val Glu Val Ile Val Arg Leu
Leu Gln Ser Phe Gly 195 200 205Trp
Val Trp Ile Ser Leu Val Gly Ser Tyr Gly Asp Tyr Gly Gln Leu 210
215 220Gly Val Gln Ala Leu Glu Glu Leu Ala Thr
Pro Arg Gly Ile Cys Val225 230 235
240Ala Phe Lys Asp Val Val Pro Leu Ser Ala Gln Ala Gly Asp Pro
Arg 245 250 255Met Gln Arg
Met Met Leu Arg Leu Ala Arg Ala Arg Thr Thr Val Val 260
265 270Val Val Phe Ser Asn Arg His Leu Ala Gly
Val Phe Phe Arg Ser Val 275 280
285Val Leu Ala Asn Leu Thr Gly Lys Val Trp Ile Ala Ser Glu Asp Trp 290
295 300Ala Ile Ser Thr Tyr Ile Thr Asn
Val Pro Gly Ile Gln Gly Ile Gly305 310
315 320Thr Val Leu Gly Val Ala Ile Gln Gln Arg Gln Val
Pro Gly Leu Lys 325 330
335Glu Phe Glu Glu Ser Tyr Val Gln Ala Val Met Gly Ala Pro Arg Thr
340 345 350Cys Pro Glu Gly Ser Trp
Cys Gly Thr Asn Gln Leu Cys Arg Glu Cys 355 360
365His Ala Phe Thr Thr Trp Asn Met Pro Glu Leu Gly Ala Phe
Ser Met 370 375 380Ser Ala Ala Tyr Asn
Val Tyr Glu Ala Val Tyr Ala Val Ala His Gly385 390
395 400Leu His Gln Leu Leu Gly Cys Thr Ser Gly
Thr Cys Ala Arg Gly Pro 405 410
415Val Tyr Pro Trp Gln Leu Leu Gln Gln Ile Tyr Lys Val Asn Phe Leu
420 425 430Leu His Lys Lys Thr
Val Ala Phe Asp Asp Lys Gly Asp Pro Leu Gly 435
440 445Tyr Tyr Asp Ile Ile Ala Trp Asp Trp Asn Gly Pro
Glu Trp Thr Phe 450 455 460Glu Val Ile
Gly Ser Ala Ser Leu Ser Pro Val His Leu Asp Ile Asn465
470 475 480Lys Thr Lys Ile Gln Trp His
Gly Lys Asn Asn Gln Val Pro Val Ser 485
490 495Val Cys Thr Arg Asp Cys Leu Glu Gly His His Arg
Leu Val Met Gly 500 505 510Ser
His His Cys Cys Phe Glu Cys Met Pro Cys Glu Ala Gly Thr Phe 515
520 525Leu Asn Thr Ser Glu Leu His Thr Cys
Gln Pro Cys Gly Thr Glu Glu 530 535
540Trp Ala Pro Glu Gly Ser Ser Ala Cys Phe Ser Arg Thr Val Glu Phe545
550 555 560Leu Gly Trp His
Glu Pro Ile Ser Leu Val Leu Leu Ala Ala Asn Thr 565
570 575Leu Leu Leu Leu Leu Leu Ile Gly Thr Ala
Gly Leu Phe Ala Trp Arg 580 585
590Leu His Thr Pro Val Val Arg Ser Ala Gly Gly Arg Leu Cys Phe Leu
595 600 605Met Leu Gly Ser Leu Val Ala
Gly Ser Cys Ser Leu Tyr Ser Phe Phe 610 615
620Gly Lys Pro Thr Val Pro Ala Cys Leu Leu Arg Gln Pro Leu Phe
Ser625 630 635 640Leu Gly
Phe Ala Ile Phe Leu Ser Cys Leu Thr Ile Arg Ser Phe Gln
645 650 655Leu Val Ile Ile Phe Lys Phe
Ser Thr Lys Val Pro Thr Phe Tyr His 660 665
670Thr Trp Ala Gln Asn His Gly Ala Gly Ile Phe Val Ile Val
Ser Ser 675 680 685Thr Val His Leu
Phe Leu Cys Leu Thr Trp Leu Ala Met Trp Thr Pro 690
695 700Arg Pro Thr Arg Glu Tyr Gln Arg Phe Pro His Leu
Val Ile Leu Glu705 710 715
720Cys Thr Glu Val Asn Ser Val Gly Phe Leu Val Ala Phe Ala His Asn
725 730 735Ile Leu Leu Ser Ile
Ser Thr Phe Val Cys Ser Tyr Leu Gly Lys Glu 740
745 750Leu Pro Glu Asn Tyr Asn Glu Ala Lys Cys Val Thr
Phe Ser Leu Leu 755 760 765Leu His
Phe Val Ser Trp Ile Ala Phe Phe Thr Met Ser Ser Ile Tyr 770
775 780Gln Gly Ser Tyr Leu Pro Ala Val Asn Val Leu
Ala Gly Leu Ala Thr785 790 795
800Leu Ser Gly Gly Phe Ser Gly Tyr Phe Leu Pro Lys Cys Tyr Val Ile
805 810 815Leu Cys Arg Pro
Glu Leu Asn Asn Thr Glu His Phe Gln Ala Ser Ile 820
825 830Gln Asp Tyr Thr Arg Arg Cys Gly Thr Thr
835 84020842PRTMus musculus 20Met Leu Phe Trp Ala Ala
His Leu Leu Leu Ser Leu Gln Leu Ala Val1 5
10 15Ala Tyr Cys Trp Ala Phe Ser Cys Gln Arg Thr Glu
Ser Ser Pro Gly 20 25 30Phe
Ser Leu Pro Gly Asp Phe Leu Leu Ala Gly Leu Phe Ser Leu His 35
40 45Ala Asp Cys Leu Gln Val Arg His Arg
Pro Leu Val Thr Ser Cys Asp 50 55
60Arg Ser Asp Ser Phe Asn Gly His Gly Tyr His Leu Phe Gln Ala Met65
70 75 80Arg Phe Thr Val Glu
Glu Ile Asn Asn Ser Thr Ala Leu Leu Pro Asn 85
90 95Ile Thr Leu Gly Tyr Glu Leu Tyr Asp Val Cys
Ser Glu Ser Ser Asn 100 105
110Val Tyr Ala Thr Leu Arg Val Leu Ala Gln Gln Gly Thr Gly His Leu
115 120 125Glu Met Gln Arg Asp Leu Arg
Asn His Ser Ser Lys Val Val Ala Leu 130 135
140Ile Gly Pro Asp Asn Thr Asp His Ala Val Thr Thr Ala Ala Leu
Leu145 150 155 160Ser Pro
Phe Leu Met Pro Leu Val Ser Tyr Glu Ala Ser Ser Val Ile
165 170 175Leu Ser Gly Lys Arg Lys Phe
Pro Ser Phe Leu Arg Thr Ile Pro Ser 180 185
190Asp Lys Tyr Gln Val Glu Val Ile Val Arg Leu Leu Gln Ser
Phe Gly 195 200 205Trp Val Trp Ile
Ser Leu Val Gly Ser Tyr Gly Asp Tyr Gly Gln Leu 210
215 220Gly Val Gln Ala Leu Glu Glu Leu Ala Thr Pro Arg
Gly Ile Cys Val225 230 235
240Ala Phe Lys Asp Val Val Pro Leu Ser Ala Gln Ala Gly Asp Pro Arg
245 250 255Met Gln Arg Met Met
Leu Arg Leu Ala Arg Ala Arg Thr Thr Val Val 260
265 270Val Val Phe Ser Asn Arg His Leu Ala Gly Val Phe
Phe Arg Ser Val 275 280 285Val Leu
Ala Asn Leu Thr Gly Lys Val Trp Ile Ala Ser Glu Asp Trp 290
295 300Ala Ile Ser Thr Tyr Ile Thr Asn Val Pro Gly
Ile Gln Gly Ile Gly305 310 315
320Thr Val Leu Gly Val Ala Ile Gln Gln Arg Gln Val Pro Gly Leu Lys
325 330 335Glu Phe Glu Glu
Ser Tyr Val Gln Ala Val Met Gly Ala Pro Arg Thr 340
345 350Cys Pro Glu Gly Ser Trp Cys Gly Thr Asn Gln
Leu Cys Arg Glu Cys 355 360 365His
Ala Phe Thr Thr Trp Asn Met Pro Glu Leu Gly Ala Phe Ser Met 370
375 380Ser Ala Ala Tyr Asn Val Tyr Glu Ala Val
Tyr Ala Val Ala His Gly385 390 395
400Leu His Gln Leu Leu Gly Cys Thr Ser Gly Thr Cys Ala Arg Gly
Pro 405 410 415Val Tyr Pro
Trp Gln Leu Leu Gln Gln Ile Tyr Lys Val Asn Phe Leu 420
425 430Leu His Lys Lys Thr Val Ala Phe Asp Asp
Lys Gly Asp Pro Leu Gly 435 440
445Tyr Tyr Asp Ile Ile Ala Trp Asp Trp Asn Gly Pro Glu Trp Thr Phe 450
455 460Glu Val Ile Gly Ser Ala Ser Leu
Ser Pro Val His Leu Asp Ile Asn465 470
475 480Lys Thr Lys Ile Gln Trp His Gly Lys Asn Asn Gln
Val Pro Val Ser 485 490
495Val Cys Thr Arg Asp Cys Leu Glu Gly His His Arg Leu Val Met Gly
500 505 510Ser His His Cys Cys Phe
Glu Cys Met Pro Cys Glu Ala Gly Thr Phe 515 520
525Leu Asn Thr Ser Glu Leu His Thr Cys Gln Pro Cys Gly Thr
Glu Glu 530 535 540Trp Ala Pro Glu Gly
Ser Ser Ala Cys Phe Ser Arg Thr Val Glu Phe545 550
555 560Leu Gly Trp His Glu Pro Ile Ser Leu Val
Leu Leu Ala Ala Asn Thr 565 570
575Leu Leu Leu Leu Leu Leu Ile Gly Thr Ala Gly Leu Phe Ala Trp Arg
580 585 590Leu His Thr Pro Val
Val Arg Ser Ala Gly Gly Arg Leu Cys Phe Leu 595
600 605Met Leu Gly Ser Leu Val Ala Gly Ser Cys Ser Leu
Tyr Ser Phe Phe 610 615 620Gly Lys Pro
Thr Val Pro Ala Cys Leu Leu Arg Gln Pro Leu Phe Ser625
630 635 640Leu Gly Phe Ala Ile Phe Leu
Ser Cys Leu Thr Ile Arg Ser Phe Gln 645
650 655Leu Val Ile Ile Phe Lys Phe Ser Thr Lys Val Pro
Thr Phe Tyr His 660 665 670Thr
Trp Ala Gln Asn His Gly Ala Gly Ile Phe Val Ile Val Ser Ser 675
680 685Thr Val His Leu Phe Leu Cys Leu Thr
Trp Leu Ala Met Trp Thr Pro 690 695
700Arg Pro Thr Arg Glu Tyr Gln Arg Phe Pro His Leu Val Ile Leu Glu705
710 715 720Cys Thr Glu Val
Asn Ser Val Gly Phe Leu Val Ala Phe Ala His Asn 725
730 735Ile Leu Leu Ser Ile Ser Thr Phe Val Cys
Ser Tyr Leu Gly Lys Glu 740 745
750Leu Pro Glu Asn Tyr Asn Glu Ala Lys Cys Val Thr Phe Ser Leu Leu
755 760 765Leu His Phe Val Ser Trp Ile
Ala Phe Phe Thr Met Ser Ser Ile Tyr 770 775
780Gln Gly Ser Tyr Leu Pro Ala Val Asn Val Leu Ala Gly Leu Ala
Thr785 790 795 800Leu Ser
Gly Gly Phe Ser Gly Tyr Phe Leu Pro Lys Cys Tyr Val Ile
805 810 815Leu Cys Arg Pro Glu Leu Asn
Asn Thr Glu His Phe Gln Ala Ser Ile 820 825
830Gln Asp Tyr Thr Arg Arg Cys Gly Thr Thr 835
84021858PRTMus musculus 21Met Pro Ala Leu Ala Ile Met Gly Leu
Ser Leu Ala Ala Phe Leu Glu1 5 10
15Leu Gly Met Gly Ala Ser Leu Cys Leu Ser Gln Gln Phe Lys Ala
Gln 20 25 30Gly Asp Tyr Ile
Leu Gly Gly Leu Phe Pro Leu Gly Ser Thr Glu Glu 35
40 45Ala Thr Leu Asn Gln Arg Ala Gln Pro Asn Ser Thr
Leu Cys Asn Arg 50 55 60Phe Ser Pro
Leu Gly Leu Phe Leu Ala Met Ala Met Lys Met Ala Val65 70
75 80Glu Glu Ile Asn Asn Gly Ser Ala
Leu Leu Pro Gly Leu Arg Leu Gly 85 90
95Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Thr Met
Lys Ser 100 105 110Ser Leu Met
Phe Leu Ala Lys Val Gly Ser Gln Ser Ile Ala Ala Tyr 115
120 125Cys Asn Tyr Thr Gln Tyr Gln Pro Arg Val Leu
Ala Val Ile Gly Pro 130 135 140His Ser
Ser Glu Leu Ala Leu Ile Thr Gly Lys Phe Phe Ser Phe Phe145
150 155 160Leu Met Pro Gln Val Ser Tyr
Ser Ala Ser Met Asp Arg Leu Ser Asp 165
170 175Arg Glu Thr Phe Pro Ser Phe Leu Arg Thr Val Pro
Ser Asp Arg Val 180 185 190Gln
Leu Gln Ala Val Val Thr Leu Leu Gln Asn Phe Ser Trp Asn Trp 195
200 205Val Ala Ala Leu Gly Ser Asp Asp Asp
Tyr Gly Arg Glu Gly Leu Ser 210 215
220Ile Phe Ser Ser Leu Ala Asn Ala Arg Gly Ile Cys Ile Ala His Glu225
230 235 240Gly Leu Val Pro
Gln His Asp Thr Ser Gly Gln Gln Leu Gly Lys Val 245
250 255Leu Asp Val Leu Arg Gln Val Asn Gln Ser
Lys Val Gln Val Val Val 260 265
270Leu Phe Ala Ser Ala Arg Ala Val Tyr Ser Leu Phe Ser Tyr Ser Ile
275 280 285His His Gly Leu Ser Pro Lys
Val Trp Val Ala Ser Glu Ser Trp Leu 290 295
300Thr Ser Asp Leu Val Met Thr Leu Pro Asn Ile Ala Arg Val Gly
Thr305 310 315 320Val Leu
Gly Phe Leu Gln Arg Gly Ala Leu Leu Pro Glu Phe Ser His
325 330 335Tyr Val Glu Thr His Leu Ala
Leu Ala Ala Asp Pro Ala Phe Cys Ala 340 345
350Ser Leu Asn Ala Glu Leu Asp Leu Glu Glu His Val Met Gly
Gln Arg 355 360 365Cys Pro Gln Cys
Asp Asp Ile Met Leu Gln Asn Leu Ser Ser Gly Leu 370
375 380Leu Gln Asn Leu Ser Ala Gly Gln Leu His His Gln
Ile Phe Ala Thr385 390 395
400Tyr Ala Ala Val Tyr Ser Val Ala Gln Ala Leu His Asn Thr Leu Gln
405 410 415Cys Asn Val Ser His
Cys His Val Ser Glu His Val Leu Pro Trp Gln 420
425 430Leu Leu Glu Asn Met Tyr Asn Met Ser Phe His Ala
Arg Asp Leu Thr 435 440 445Leu Gln
Phe Asp Ala Glu Gly Asn Val Asp Met Glu Tyr Asp Leu Lys 450
455 460Met Trp Val Trp Gln Ser Pro Thr Pro Val Leu
His Thr Val Gly Thr465 470 475
480Phe Asn Gly Thr Leu Gln Leu Gln Gln Ser Lys Met Tyr Trp Pro Gly
485 490 495Asn Gln Val Pro
Val Ser Gln Cys Ser Arg Gln Cys Lys Asp Gly Gln 500
505 510Val Arg Arg Val Lys Gly Phe His Ser Cys Cys
Tyr Asp Cys Val Asp 515 520 525Cys
Lys Ala Gly Ser Tyr Arg Lys His Pro Asp Asp Phe Thr Cys Thr 530
535 540Pro Cys Asn Gln Asp Gln Trp Ser Pro Glu
Lys Ser Thr Ala Cys Leu545 550 555
560Pro Arg Arg Pro Lys Phe Leu Ala Trp Gly Glu Pro Val Val Leu
Ser 565 570 575Leu Leu Leu
Leu Leu Cys Leu Val Leu Gly Leu Ala Leu Ala Ala Leu 580
585 590Gly Leu Ser Val His His Trp Asp Ser Pro
Leu Val Gln Ala Ser Gly 595 600
605Gly Ser Gln Phe Cys Phe Gly Leu Ile Cys Leu Gly Leu Phe Cys Leu 610
615 620Ser Val Leu Leu Phe Pro Gly Arg
Pro Ser Ser Ala Ser Cys Leu Ala625 630
635 640Gln Gln Pro Met Ala His Leu Pro Leu Thr Gly Cys
Leu Ser Thr Leu 645 650
655Phe Leu Gln Ala Ala Glu Thr Phe Val Glu Ser Glu Leu Pro Leu Ser
660 665 670Trp Ala Asn Trp Leu Cys
Ser Tyr Leu Arg Gly Leu Trp Ala Trp Leu 675 680
685Val Val Leu Leu Ala Thr Phe Val Glu Ala Ala Leu Cys Ala
Trp Tyr 690 695 700Leu Thr Ala Phe Pro
Pro Glu Val Val Thr Asp Trp Ser Val Leu Pro705 710
715 720Thr Glu Val Leu Glu His Cys His Val Arg
Ser Trp Val Ser Leu Gly 725 730
735Leu Val His Ile Thr Asn Ala Met Leu Ala Phe Leu Cys Phe Leu Gly
740 745 750Thr Phe Leu Val Gln
Ser Gln Pro Gly Arg Tyr Asn Arg Ala Arg Gly 755
760 765Leu Thr Phe Ala Met Leu Ala Tyr Phe Ile Thr Trp
Val Ser Phe Val 770 775 780Pro Leu Leu
Ala Asn Val Gln Val Ala Tyr Gln Pro Ala Val Gln Met785
790 795 800Gly Ala Ile Leu Val Cys Ala
Leu Gly Ile Leu Val Thr Phe His Leu 805
810 815Pro Lys Cys Tyr Val Leu Leu Trp Leu Pro Lys Leu
Asn Thr Gln Glu 820 825 830Phe
Phe Leu Gly Arg Asn Ala Lys Lys Ala Ala Asp Glu Asn Ser Gly 835
840 845Gly Gly Glu Ala Ala Gln Glu His Asn
Glu 850 85522842PRTRattus norvegicus 22Met Leu Phe Trp
Ala Ala His Leu Leu Leu Ser Leu Gln Leu Ala Val1 5
10 15Ala Tyr Cys Trp Ala Phe Ser Cys Gln Arg
Thr Glu Ser Ser Pro Gly 20 25
30Phe Ser Leu Pro Gly Asp Phe Leu Leu Ala Gly Leu Phe Ser Leu His
35 40 45Ala Asp Cys Leu Gln Val Arg His
Arg Pro Leu Val Thr Ser Cys Asp 50 55
60Arg Ser Asp Ser Phe Asn Gly His Gly Tyr His Leu Phe Gln Ala Met65
70 75 80Arg Phe Thr Val Glu
Glu Ile Asn Asn Ser Thr Ala Leu Leu Pro Asn 85
90 95Ile Thr Leu Gly Tyr Glu Leu Tyr Asp Val Cys
Ser Glu Ser Ser Asn 100 105
110Val Tyr Ala Thr Leu Arg Val Leu Ala Gln Gln Gly Thr Gly His Leu
115 120 125Glu Met Gln Arg Asp Leu Arg
Asn His Ser Ser Lys Val Val Ala Leu 130 135
140Ile Gly Pro Asp Asn Thr Asp His Ala Val Thr Thr Ala Ala Leu
Leu145 150 155 160Ser Pro
Phe Leu Met Pro Leu Val Ser Tyr Glu Ala Ser Ser Val Ile
165 170 175Leu Ser Gly Lys Arg Lys Phe
Pro Ser Phe Leu Arg Thr Ile Pro Ser 180 185
190Asp Lys Tyr Gln Val Glu Val Ile Val Arg Leu Leu Gln Ser
Phe Gly 195 200 205Trp Val Trp Ile
Ser Leu Val Gly Ser Tyr Gly Asp Tyr Gly Gln Leu 210
215 220Gly Val Gln Ala Leu Glu Glu Leu Ala Thr Pro Arg
Gly Ile Cys Val225 230 235
240Ala Phe Lys Asp Val Val Pro Leu Ser Ala Gln Ala Gly Asp Pro Arg
245 250 255Met Gln Arg Met Met
Leu Arg Leu Ala Arg Ala Arg Thr Thr Val Val 260
265 270Val Val Phe Ser Asn Arg His Leu Ala Gly Val Phe
Phe Arg Ser Val 275 280 285Val Leu
Ala Asn Leu Thr Gly Lys Val Trp Ile Ala Ser Glu Asp Trp 290
295 300Ala Ile Ser Thr Tyr Ile Thr Asn Val Pro Gly
Ile Gln Gly Ile Gly305 310 315
320Thr Val Leu Gly Val Ala Ile Gln Gln Arg Gln Val Pro Gly Leu Lys
325 330 335Glu Phe Glu Glu
Ser Tyr Val Gln Ala Val Thr Gly Ala Pro Arg Thr 340
345 350Cys Pro Glu Gly Ser Trp Cys Gly Thr Asn Gln
Leu Cys Arg Glu Cys 355 360 365His
Ala Phe Thr Thr Trp Asn Met Pro Glu Leu Gly Ala Phe Ser Met 370
375 380Ser Ala Ala Tyr Asn Val Tyr Glu Ala Val
Tyr Ala Val Ala His Gly385 390 395
400Leu His Gln Leu Leu Gly Cys Thr Ser Gly Thr Cys Ala Arg Gly
Pro 405 410 415Val Tyr Pro
Trp Gln Leu Leu Gln Gln Ile Tyr Lys Val Asn Phe Leu 420
425 430Leu His Lys Lys Thr Val Ala Phe Asp Asp
Asn Gly Asp Pro Leu Gly 435 440
445Tyr Tyr Asp Ile Ile Ala Trp Asp Trp Asn Gly Pro Glu Trp Thr Phe 450
455 460Glu Val Ile Gly Ser Ala Ser Leu
Ser Pro Val His Leu Asp Ile Asn465 470
475 480Lys Thr Lys Ile Gln Trp His Gly Lys Asn Asn Gln
Val Pro Val Ser 485 490
495Val Cys Thr Arg Asp Cys Leu Glu Gly His His Arg Leu Val Met Gly
500 505 510Ser His His Cys Cys Phe
Glu Cys Met Pro Cys Glu Ala Gly Thr Phe 515 520
525Leu Asn Thr Ser Glu Leu His Thr Cys Gln Pro Cys Gly Thr
Glu Glu 530 535 540Trp Ala Pro Glu Gly
Ser Ser Ala Cys Phe Ser Arg Thr Val Glu Phe545 550
555 560Leu Gly Trp His Glu Pro Ile Ser Leu Val
Leu Leu Ala Ala Asn Thr 565 570
575Leu Leu Leu Leu Leu Leu Ile Gly Thr Ala Gly Leu Phe Ala Trp Arg
580 585 590Leu His Thr Pro Val
Val Arg Ser Ala Gly Gly Arg Leu Cys Phe Leu 595
600 605Met Leu Gly Ser Leu Val Ala Gly Ser Cys Ser Leu
Tyr Ser Phe Phe 610 615 620Gly Glu Pro
Thr Val Pro Ala Cys Leu Leu Arg Gln Pro Leu Phe Ser625
630 635 640Leu Gly Phe Ala Ile Phe Leu
Ser Cys Leu Thr Ile Arg Ser Phe Gln 645
650 655Leu Val Ile Ile Phe Lys Phe Ser Thr Lys Val Pro
Thr Phe Tyr His 660 665 670Thr
Trp Ala Gln Asn His Gly Ala Gly Ile Phe Val Ile Val Ser Ser 675
680 685Thr Val His Leu Phe Leu Cys Leu Thr
Trp Leu Ala Met Trp Thr Pro 690 695
700Arg Pro Thr Arg Glu Tyr Gln Arg Phe Pro His Leu Val Ile Leu Glu705
710 715 720Cys Thr Glu Val
Asn Ser Val Gly Phe Leu Val Ala Phe Ala His Asn 725
730 735Ile Leu Leu Ser Ile Ser Thr Phe Val Cys
Ser Tyr Leu Gly Lys Glu 740 745
750Leu Pro Glu Asn Tyr Asn Glu Ala Lys Cys Val Thr Phe Ser Leu Leu
755 760 765Leu His Phe Val Ser Trp Ile
Ala Phe Phe Thr Met Ser Ser Ile Tyr 770 775
780Gln Gly Ser Tyr Leu Pro Ala Val Asn Val Leu Ala Gly Leu Ala
Thr785 790 795 800Leu Ser
Gly Gly Phe Ser Gly Tyr Phe Leu Pro Lys Cys Tyr Val Ile
805 810 815Leu Cys Arg Pro Glu Leu Asn
Asn Thr Glu His Phe Gln Ala Ser Ile 820 825
830Gln Asp Tyr Thr Arg Arg Cys Gly Thr Thr 835
84023842PRTMus musculus 23Met Leu Phe Trp Ala Ala His Leu Leu
Leu Ser Leu Gln Leu Ala Val1 5 10
15Ala Tyr Cys Trp Ala Phe Ser Cys Gln Arg Thr Glu Ser Ser Pro
Gly 20 25 30Phe Ser Leu Pro
Gly Asp Phe Leu Leu Ala Gly Leu Phe Ser Leu His 35
40 45Ala Asp Cys Leu Gln Val Arg His Arg Pro Leu Val
Thr Ser Cys Asp 50 55 60Arg Ser Asp
Ser Phe Asn Gly His Gly Tyr His Leu Phe Gln Ala Met65 70
75 80Arg Phe Thr Val Glu Glu Ile Asn
Asn Ser Thr Ala Leu Leu Pro Asn 85 90
95Ile Thr Leu Gly Tyr Glu Leu Tyr Asp Val Cys Ser Glu Ser
Ser Asn 100 105 110Val Tyr Ala
Thr Leu Arg Val Leu Ala Gln Gln Gly Thr Gly His Leu 115
120 125Glu Met Gln Arg Asp Leu Arg Asn His Ser Ser
Lys Val Val Ala Leu 130 135 140Ile Gly
Pro Asp Asn Thr Asp His Ala Val Thr Thr Ala Ala Leu Leu145
150 155 160Ser Pro Phe Leu Met Pro Leu
Val Ser Tyr Glu Ala Ser Ser Val Ile 165
170 175Leu Ser Gly Lys Arg Lys Phe Pro Ser Phe Leu Arg
Thr Ile Pro Ser 180 185 190Asp
Lys Tyr Gln Val Glu Val Ile Val Arg Leu Leu Gln Ser Phe Gly 195
200 205Trp Val Trp Ile Ser Leu Val Gly Ser
Tyr Gly Asp Tyr Gly Gln Leu 210 215
220Gly Val Gln Ala Leu Glu Glu Leu Ala Thr Pro Arg Gly Ile Cys Val225
230 235 240Ala Phe Lys Asp
Val Val Pro Leu Ser Ala Gln Ala Gly Asp Pro Arg 245
250 255Met Gln Arg Met Met Leu Arg Leu Ala Arg
Ala Arg Thr Thr Val Val 260 265
270Val Val Phe Ser Asn Arg His Leu Ala Gly Val Phe Phe Arg Ser Val
275 280 285Val Leu Ala Asn Leu Thr Gly
Lys Val Trp Ile Ala Ser Glu Asp Trp 290 295
300Ala Ile Ser Thr Tyr Ile Thr Asn Val Pro Gly Ile Gln Gly Ile
Gly305 310 315 320Thr Val
Leu Gly Val Ala Ile Gln Gln Arg Gln Val Pro Gly Leu Lys
325 330 335Glu Phe Glu Glu Ser Tyr Val
Gln Ala Val Met Gly Ala Pro Arg Thr 340 345
350Cys Pro Glu Gly Ser Trp Cys Gly Thr Asn Gln Leu Cys Arg
Glu Cys 355 360 365His Ala Phe Thr
Thr Trp Asn Met Pro Glu Leu Gly Ala Phe Ser Met 370
375 380Ser Ala Ala Tyr Asn Val Tyr Glu Ala Val Tyr Ala
Val Ala His Gly385 390 395
400Leu His Gln Leu Leu Gly Cys Thr Ser Gly Thr Cys Ala Arg Gly Pro
405 410 415Val Tyr Pro Trp Gln
Leu Leu Gln Gln Ile Tyr Lys Val Asn Phe Leu 420
425 430Leu His Lys Lys Thr Val Ala Phe Asp Asp Lys Gly
Asp Pro Leu Gly 435 440 445Tyr Tyr
Asp Ile Ile Ala Trp Asp Trp Asn Gly Pro Glu Trp Thr Phe 450
455 460Glu Val Ile Gly Ser Ala Ser Leu Ser Pro Val
His Leu Asp Ile Asn465 470 475
480Lys Thr Lys Ile Gln Trp His Gly Lys Asn Asn Gln Val Pro Val Ser
485 490 495Val Cys Thr Arg
Asp Cys Leu Glu Gly His His Arg Leu Val Met Gly 500
505 510Ser His His Cys Cys Phe Glu Cys Met Pro Cys
Glu Ala Gly Thr Phe 515 520 525Leu
Asn Thr Ser Glu Leu His Thr Cys Gln Pro Cys Gly Thr Glu Glu 530
535 540Trp Ala Pro Glu Gly Ser Ser Ala Cys Phe
Ser Arg Thr Val Glu Phe545 550 555
560Leu Gly Trp His Glu Pro Ile Ser Leu Val Leu Leu Ala Ala Asn
Thr 565 570 575Leu Leu Leu
Leu Leu Leu Ile Gly Thr Ala Gly Leu Phe Ala Trp Arg 580
585 590Leu His Thr Pro Val Val Arg Ser Ala Gly
Gly Arg Leu Cys Phe Leu 595 600
605Met Leu Gly Ser Leu Val Ala Gly Ser Cys Ser Leu Tyr Ser Phe Phe 610
615 620Gly Lys Pro Thr Val Pro Ala Cys
Leu Leu Arg Gln Pro Leu Phe Ser625 630
635 640Leu Gly Phe Ala Ile Phe Leu Ser Cys Leu Thr Ile
Arg Ser Phe Gln 645 650
655Leu Val Ile Ile Phe Lys Phe Ser Thr Lys Val Pro Thr Phe Tyr His
660 665 670Thr Trp Ala Gln Asn His
Gly Ala Gly Ile Phe Val Ile Val Ser Ser 675 680
685Thr Val His Leu Phe Leu Cys Leu Thr Trp Leu Ala Met Trp
Thr Pro 690 695 700Arg Pro Thr Arg Glu
Tyr Gln Arg Phe Pro His Leu Val Ile Leu Glu705 710
715 720Cys Thr Glu Val Asn Ser Val Gly Phe Leu
Val Ala Phe Ala His Asn 725 730
735Ile Leu Leu Ser Ile Ser Thr Phe Val Cys Ser Tyr Leu Gly Lys Glu
740 745 750Leu Pro Glu Asn Tyr
Asn Glu Ala Lys Cys Val Thr Phe Ser Leu Leu 755
760 765Leu His Phe Val Ser Trp Ile Ala Phe Phe Thr Met
Ser Ser Ile Tyr 770 775 780Gln Gly Ser
Tyr Leu Pro Ala Val Asn Val Leu Ala Gly Leu Ala Thr785
790 795 800Leu Ser Gly Gly Phe Ser Gly
Tyr Phe Leu Pro Lys Cys Tyr Val Ile 805
810 815Leu Cys Arg Pro Glu Leu Asn Asn Thr Glu His Phe
Gln Ala Ser Ile 820 825 830Gln
Asp Tyr Thr Arg Arg Cys Gly Thr Thr 835
84024842PRTMus musculus 24Met Leu Phe Trp Ala Ala His Leu Leu Leu Ser Leu
Gln Leu Ala Val1 5 10
15Ala Tyr Cys Trp Ala Phe Ser Cys Gln Arg Thr Glu Ser Ser Pro Gly
20 25 30Phe Ser Leu Pro Gly Asp Phe
Leu Leu Ala Gly Leu Phe Ser Leu His 35 40
45Ala Asp Cys Leu Gln Val Arg His Arg Pro Leu Val Thr Ser Cys
Asp 50 55 60Arg Ser Asp Ser Phe Asn
Gly His Gly Tyr His Leu Phe Gln Ala Met65 70
75 80Arg Phe Thr Val Glu Glu Ile Asn Asn Ser Thr
Ala Leu Leu Pro Asn 85 90
95Ile Thr Leu Gly Tyr Glu Leu Tyr Asp Val Cys Ser Glu Ser Ser Asn
100 105 110Val Tyr Ala Thr Leu Arg
Val Leu Ala Gln Gln Gly Thr Gly His Leu 115 120
125Glu Met Gln Arg Asp Leu Arg Asn His Ser Ser Lys Val Val
Ala Leu 130 135 140Ile Gly Pro Asp Asn
Thr Asp His Ala Val Thr Thr Ala Ala Leu Leu145 150
155 160Ser Pro Phe Leu Met Pro Leu Val Ser Tyr
Glu Ala Ser Ser Val Ile 165 170
175Leu Ser Gly Lys Arg Lys Phe Pro Ser Phe Leu Arg Thr Ile Pro Ser
180 185 190Asp Lys Tyr Gln Val
Glu Val Ile Val Arg Leu Leu Gln Ser Phe Gly 195
200 205Trp Val Trp Ile Ser Leu Val Gly Ser Tyr Gly Asp
Tyr Gly Gln Leu 210 215 220Gly Val Gln
Ala Leu Glu Glu Leu Ala Thr Pro Arg Gly Ile Cys Val225
230 235 240Ala Phe Lys Asn Val Val Pro
Leu Ser Ala Gln Ala Gly Asp Pro Arg 245
250 255Met Gln Arg Met Met Leu Arg Leu Ala Arg Ala Arg
Thr Thr Val Val 260 265 270Val
Val Phe Ser Asn Arg His Leu Asp Gly Val Phe Phe Arg Ser Val 275
280 285Val Leu Ala Asn Leu Thr Gly Lys Val
Trp Ile Ala Ser Glu Asp Trp 290 295
300Ala Ile Ser Thr Asn Ile Pro Asn Val Ser Gly Ile Gln Gly Ile Gly305
310 315 320Thr Val Leu Gly
Val Ala Ile Gln Gln Arg Gln Val Pro Gly Leu Lys 325
330 335Glu Phe Glu Glu Ser Tyr Val Gln Ala Val
Met Gly Ala Pro Arg Thr 340 345
350Cys Pro Glu Gly Ser Trp Cys Gly Thr Asn Gln Leu Cys Arg Glu Cys
355 360 365His Ala Phe Thr Thr Trp Asn
Met Pro Glu Leu Gly Ala Phe Ser Met 370 375
380Ser Ala Ala Tyr Asn Val Tyr Glu Ala Val Tyr Ala Val Ala His
Gly385 390 395 400Leu His
Gln Leu Leu Gly Cys Thr Ser Gly Thr Cys Ala Arg Gly Pro
405 410 415Val His Pro Trp Gln Leu Leu
Gln Gln Ile Tyr Lys Val Asn Phe Leu 420 425
430Leu His Lys Lys Thr Val Ala Phe Asp Asp Lys Gly Asp Pro
Leu Gly 435 440 445Tyr Tyr Asp Ile
Ile Ala Trp Asp Trp Asn Gly Pro Glu Trp Thr Phe 450
455 460Glu Val Ile Gly Ser Ala Ser Leu Ser Pro Val His
Leu Asp Ile Asn465 470 475
480Lys Thr Lys Ile Gln Trp His Gly Lys Asn Asn Gln Val Pro Val Ser
485 490 495Val Cys Thr Arg Asp
Cys Leu Glu Gly His His Arg Leu Val Met Gly 500
505 510Ser His His Cys Cys Phe Glu Cys Met Pro Cys Glu
Ala Gly Thr Phe 515 520 525Leu Asn
Thr Ser Glu Leu His Thr Cys Gln Pro Cys Gly Thr Glu Glu 530
535 540Trp Ala Pro Glu Gly Ser Ser Ala Cys Phe Ser
Arg Thr Val Glu Phe545 550 555
560Leu Gly Trp His Glu Pro Ile Ser Leu Val Leu Leu Ala Ala Asn Thr
565 570 575Leu Leu Leu Leu
Leu Leu Ile Gly Thr Ala Gly Leu Phe Ala Trp Arg 580
585 590Leu His Thr Pro Val Val Arg Ser Ala Gly Gly
Arg Leu Cys Phe Leu 595 600 605Met
Leu Gly Ser Leu Val Ala Gly Ser Cys Ser Leu Tyr Ser Phe Phe 610
615 620Gly Lys Pro Thr Val Pro Ala Cys Leu Leu
Arg Gln Pro Leu Phe Ser625 630 635
640Leu Gly Phe Ala Ile Phe Leu Ser Cys Leu Thr Ile Arg Ser Phe
Gln 645 650 655Leu Val Ile
Ile Phe Lys Phe Ser Thr Lys Val Pro Thr Phe Tyr His 660
665 670Thr Trp Ala Gln Asn His Gly Ala Gly Ile
Phe Val Ile Val Ser Ser 675 680
685Thr Val His Leu Phe Leu Cys Leu Thr Trp Leu Ala Met Trp Thr Pro 690
695 700Arg Pro Thr Arg Glu Tyr Gln Arg
Phe Pro His Leu Val Ile Leu Glu705 710
715 720Cys Thr Glu Val Asn Ser Val Gly Phe Leu Val Ala
Phe Ala His Asn 725 730
735Ile Leu Leu Ser Ile Ser Thr Phe Val Cys Ser Tyr Leu Gly Lys Glu
740 745 750Leu Pro Glu Asn Tyr Asn
Glu Ala Lys Cys Val Thr Phe Ser Leu Leu 755 760
765Leu His Phe Val Ser Trp Ile Ala Phe Phe Thr Met Ser Ser
Ile Tyr 770 775 780Gln Gly Ser Tyr Leu
Pro Ala Val Asn Val Leu Ala Gly Leu Ala Thr785 790
795 800Leu Ser Gly Gly Phe Ser Gly Tyr Phe Leu
Pro Lys Cys Tyr Val Ile 805 810
815Leu Cys Arg Pro Glu Leu Asn Asn Thr Glu His Phe Gln Ala Ser Ile
820 825 830Gln Asp Tyr Thr Arg
Arg Cys Gly Thr Thr 835 84025843PRTRattus
norvegicus 25Met Gly Pro Gln Ala Arg Thr Leu Cys Leu Leu Ser Leu Leu Leu
His1 5 10 15Val Leu Pro
Lys Pro Gly Lys Leu Val Glu Asn Ser Asp Phe His Leu 20
25 30Ala Gly Asp Tyr Leu Leu Gly Gly Leu Phe
Thr Leu His Ala Asn Val 35 40
45Lys Ser Ile Ser His Leu Ser Tyr Leu Gln Val Pro Lys Cys Asn Glu 50
55 60Phe Thr Met Lys Val Leu Gly Tyr Asn
Leu Met Gln Ala Met Arg Phe65 70 75
80Ala Val Glu Glu Ile Asn Asn Cys Ser Ser Leu Leu Pro Gly
Val Leu 85 90 95Leu Gly
Tyr Glu Met Val Asp Val Cys Tyr Leu Ser Asn Asn Ile His 100
105 110Pro Gly Leu Tyr Phe Leu Ala Gln Asp
Asp Asp Leu Leu Pro Ile Leu 115 120
125Lys Asp Tyr Ser Gln Tyr Met Pro His Val Val Ala Val Ile Gly Pro
130 135 140Asp Asn Ser Glu Ser Ala Ile
Thr Val Ser Asn Ile Leu Ser His Phe145 150
155 160Leu Ile Pro Gln Ile Thr Tyr Ser Ala Ile Ser Asp
Lys Leu Arg Asp 165 170
175Lys Arg His Phe Pro Ser Met Leu Arg Thr Val Pro Ser Ala Thr His
180 185 190His Ile Glu Ala Met Val
Gln Leu Met Val His Phe Gln Trp Asn Trp 195 200
205Ile Val Val Leu Val Ser Asp Asp Asp Tyr Gly Arg Glu Asn
Ser His 210 215 220Leu Leu Ser Gln Arg
Leu Thr Lys Thr Ser Asp Ile Cys Ile Ala Phe225 230
235 240Gln Glu Val Leu Pro Ile Pro Glu Ser Ser
Gln Val Met Arg Ser Glu 245 250
255Glu Gln Arg Gln Leu Asp Asn Ile Leu Asp Lys Leu Arg Arg Thr Ser
260 265 270Ala Arg Val Val Val
Val Phe Ser Pro Glu Leu Ser Leu Tyr Ser Phe 275
280 285Phe His Glu Val Leu Arg Trp Asn Phe Thr Gly Phe
Val Trp Ile Ala 290 295 300Ser Glu Ser
Trp Ala Ile Asp Pro Val Leu His Asn Leu Thr Glu Leu305
310 315 320Arg His Thr Gly Thr Phe Leu
Gly Val Thr Ile Gln Arg Val Ser Ile 325
330 335Pro Gly Phe Ser Gln Phe Arg Val Arg Arg Asp Lys
Pro Gly Tyr Pro 340 345 350Val
Pro Asn Thr Thr Asn Leu Arg Thr Thr Cys Asn Gln Asp Cys Asp 355
360 365Ala Cys Leu Asn Thr Thr Lys Ser Phe
Asn Asn Ile Leu Ile Leu Ser 370 375
380Gly Glu Arg Val Val Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala385
390 395 400His Ala Leu His
Arg Leu Leu Gly Cys Asn Arg Val Arg Cys Thr Lys 405
410 415Gln Lys Val Tyr Pro Trp Gln Leu Leu Arg
Glu Ile Trp His Val Asn 420 425
430Phe Thr Leu Leu Gly Asn Arg Leu Phe Phe Asp Gln Gln Gly Asp Met
435 440 445Pro Met Leu Leu Asp Ile Ile
Gln Trp Gln Trp Asp Leu Ser Gln Asn 450 455
460Pro Phe Gln Ser Ile Ala Ser Tyr Ser Pro Thr Ser Lys Arg Leu
Thr465 470 475 480Tyr Ile
Asn Asn Val Ser Trp Tyr Thr Pro Asn Asn Thr Val Pro Val
485 490 495Ser Met Cys Ser Lys Ser Cys
Gln Pro Gly Gln Met Lys Lys Ser Val 500 505
510Gly Leu His Pro Cys Cys Phe Glu Cys Leu Asp Cys Met Pro
Gly Thr 515 520 525Tyr Leu Asn Arg
Ser Ala Asp Glu Phe Asn Cys Leu Ser Cys Pro Gly 530
535 540Ser Met Trp Ser Tyr Lys Asn Asp Ile Thr Cys Phe
Gln Arg Arg Pro545 550 555
560Thr Phe Leu Glu Trp His Glu Val Pro Thr Ile Val Val Ala Ile Leu
565 570 575Ala Ala Leu Gly Phe
Phe Ser Thr Leu Ala Ile Leu Phe Ile Phe Trp 580
585 590Arg His Phe Gln Thr Pro Met Val Arg Ser Ala Gly
Gly Pro Met Cys 595 600 605Phe Leu
Met Leu Val Pro Leu Leu Leu Ala Phe Gly Met Val Pro Val 610
615 620Tyr Val Gly Pro Pro Thr Val Phe Ser Cys Phe
Cys Arg Gln Ala Phe625 630 635
640Phe Thr Val Cys Phe Ser Ile Cys Leu Ser Cys Ile Thr Val Arg Ser
645 650 655Phe Gln Ile Val
Cys Val Phe Lys Met Ala Arg Arg Leu Pro Ser Ala 660
665 670Tyr Ser Phe Trp Met Arg Tyr His Gly Pro Tyr
Val Phe Val Ala Phe 675 680 685Ile
Thr Ala Ile Lys Val Ala Leu Val Val Gly Asn Met Leu Ala Thr 690
695 700Thr Ile Asn Pro Ile Gly Arg Thr Asp Pro
Asp Asp Pro Asn Ile Met705 710 715
720Ile Leu Ser Cys His Pro Asn Tyr Arg Asn Gly Leu Leu Phe Asn
Thr 725 730 735Ser Met Asp
Leu Leu Leu Ser Val Leu Gly Phe Ser Phe Ala Tyr Met 740
745 750Gly Lys Glu Leu Pro Thr Asn Tyr Asn Glu
Ala Lys Phe Ile Thr Leu 755 760
765Ser Met Thr Phe Ser Phe Thr Ser Ser Ile Ser Leu Cys Thr Phe Met 770
775 780Ser Val His Asp Gly Val Leu Val
Thr Ile Met Asp Leu Leu Val Thr785 790
795 800Val Leu Asn Phe Leu Ala Ile Gly Leu Gly Tyr Phe
Gly Pro Lys Cys 805 810
815Tyr Met Ile Leu Phe Tyr Pro Glu Arg Asn Thr Ser Ala Tyr Phe Asn
820 825 830Ser Met Ile Gln Gly Tyr
Thr Met Arg Lys Ser 835 84026843PRTMus musculus
26Met Gly Pro Gln Ala Arg Thr Leu His Leu Leu Phe Leu Leu Leu His1
5 10 15Ala Leu Pro Lys Pro Val
Met Leu Val Gly Asn Ser Asp Phe His Leu 20 25
30Ala Gly Asp Tyr Leu Leu Gly Gly Leu Phe Thr Leu His
Ala Asn Val 35 40 45Lys Ser Val
Ser His Leu Ser Tyr Leu Gln Val Pro Lys Cys Asn Glu 50
55 60Tyr Asn Met Lys Val Leu Gly Tyr Asn Leu Met Gln
Ala Met Arg Phe65 70 75
80Ala Val Glu Glu Ile Asn Asn Cys Ser Ser Leu Leu Pro Gly Val Leu
85 90 95Leu Gly Tyr Glu Met Val
Asp Val Cys Tyr Leu Ser Asn Asn Ile Gln 100
105 110Pro Gly Leu Tyr Phe Leu Ser Gln Ile Asp Asp Phe
Leu Pro Ile Leu 115 120 125Lys Asp
Tyr Ser Gln Tyr Arg Pro Gln Val Val Ala Val Ile Gly Pro 130
135 140Asp Asn Ser Glu Ser Ala Ile Thr Val Ser Asn
Ile Leu Ser Tyr Phe145 150 155
160Leu Val Pro Gln Val Thr Tyr Ser Ala Ile Thr Asp Lys Leu Arg Asp
165 170 175Lys Arg Arg Phe
Pro Ala Met Leu Arg Thr Val Pro Ser Ala Thr His 180
185 190His Ile Glu Ala Met Val Gln Leu Met Val His
Phe Gln Trp Asn Trp 195 200 205Ile
Val Val Leu Val Ser Asp Asp Asp Tyr Gly Arg Glu Asn Ser His 210
215 220Leu Leu Ser Gln Arg Leu Thr Asn Thr Gly
Asp Ile Cys Ile Ala Phe225 230 235
240Gln Glu Val Leu Pro Val Pro Glu Pro Asn Gln Ala Val Arg Pro
Glu 245 250 255Glu Gln Asp
Gln Leu Asp Asn Ile Leu Asp Lys Leu Arg Arg Thr Ser 260
265 270Ala Arg Val Val Val Ile Phe Ser Pro Glu
Leu Ser Leu His Asn Phe 275 280
285Phe Arg Glu Val Leu Arg Trp Asn Phe Thr Gly Phe Val Trp Ile Ala 290
295 300Ser Glu Ser Trp Ala Ile Asp Pro
Val Leu His Asn Leu Thr Glu Leu305 310
315 320Arg His Thr Gly Thr Phe Leu Gly Val Thr Ile Gln
Arg Val Ser Ile 325 330
335Pro Gly Phe Ser Gln Phe Arg Val Arg His Asp Lys Pro Glu Tyr Pro
340 345 350Met Pro Asn Glu Thr Ser
Leu Arg Thr Thr Cys Asn Gln Asp Cys Asp 355 360
365Ala Cys Met Asn Ile Thr Glu Ser Phe Asn Asn Val Leu Met
Leu Ser 370 375 380Gly Glu Arg Val Val
Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala385 390
395 400His Thr Leu His Arg Leu Leu His Cys Asn
Gln Val Arg Cys Thr Lys 405 410
415Gln Ile Val Tyr Pro Trp Gln Leu Leu Arg Glu Ile Trp His Val Asn
420 425 430Phe Thr Leu Leu Gly
Asn Gln Leu Phe Phe Asp Glu Gln Gly Asp Met 435
440 445Pro Met Leu Leu Asp Ile Ile Gln Trp Gln Trp Gly
Leu Ser Gln Asn 450 455 460Pro Phe Gln
Ser Ile Ala Ser Tyr Ser Pro Thr Glu Thr Arg Leu Thr465
470 475 480Tyr Ile Ser Asn Val Ser Trp
Tyr Thr Pro Asn Asn Thr Val Pro Ile 485
490 495Ser Met Cys Ser Lys Ser Cys Gln Pro Gly Gln Met
Lys Lys Pro Ile 500 505 510Gly
Leu His Pro Cys Cys Phe Glu Cys Val Asp Cys Pro Pro Gly Thr 515
520 525Tyr Leu Asn Arg Ser Val Asp Glu Phe
Asn Cys Leu Ser Cys Pro Gly 530 535
540Ser Met Trp Ser Tyr Lys Asn Asn Ile Ala Cys Phe Lys Arg Arg Leu545
550 555 560Ala Phe Leu Glu
Trp His Glu Val Pro Thr Ile Val Val Thr Ile Leu 565
570 575Ala Ala Leu Gly Phe Ile Ser Thr Leu Ala
Ile Leu Leu Ile Phe Trp 580 585
590Arg His Phe Gln Thr Pro Met Val Arg Ser Ala Gly Gly Pro Met Cys
595 600 605Phe Leu Met Leu Val Pro Leu
Leu Leu Ala Phe Gly Met Val Pro Val 610 615
620Tyr Val Gly Pro Pro Thr Val Phe Ser Cys Phe Cys Arg Gln Ala
Phe625 630 635 640Phe Thr
Val Cys Phe Ser Val Cys Leu Ser Cys Ile Thr Val Arg Ser
645 650 655Phe Gln Ile Val Cys Val Phe
Lys Met Ala Arg Arg Leu Pro Ser Ala 660 665
670Tyr Gly Phe Trp Met Arg Tyr His Gly Pro Tyr Val Phe Val
Ala Phe 675 680 685Ile Thr Ala Val
Lys Val Ala Leu Val Ala Gly Asn Met Leu Ala Thr 690
695 700Thr Ile Asn Pro Ile Gly Arg Thr Asp Pro Asp Asp
Pro Asn Ile Ile705 710 715
720Ile Leu Ser Cys His Pro Asn Tyr Arg Asn Gly Leu Leu Phe Asn Thr
725 730 735Ser Met Asp Leu Leu
Leu Ser Val Leu Gly Phe Ser Phe Ala Tyr Val 740
745 750Gly Lys Glu Leu Pro Thr Asn Tyr Asn Glu Ala Lys
Phe Ile Thr Leu 755 760 765Ser Met
Thr Phe Ser Phe Thr Ser Ser Ile Ser Leu Cys Thr Phe Met 770
775 780Ser Val His Asp Gly Val Leu Val Thr Ile Met
Asp Leu Leu Val Thr785 790 795
800Val Leu Asn Phe Leu Ala Ile Gly Leu Gly Tyr Phe Gly Pro Lys Cys
805 810 815Tyr Met Ile Leu
Phe Tyr Pro Glu Arg Asn Thr Ser Ala Tyr Phe Asn 820
825 830Ser Met Ile Gln Gly Tyr Thr Met Arg Lys Ser
835 84027858PRTMus musculus 27Met Pro Ala Leu Ala
Ile Met Gly Leu Ser Leu Ala Ala Phe Leu Glu1 5
10 15Leu Gly Met Gly Ala Ser Leu Cys Leu Ser Gln
Gln Phe Lys Ala Gln 20 25
30Gly Asp Tyr Ile Leu Gly Gly Leu Phe Pro Leu Gly Ser Thr Glu Glu
35 40 45Ala Thr Leu Asn Gln Arg Thr Gln
Pro Asn Ser Ile Pro Cys Asn Arg 50 55
60Phe Ser Pro Leu Gly Leu Phe Leu Ala Met Ala Met Lys Met Ala Val65
70 75 80Glu Glu Ile Asn Asn
Gly Ser Ala Leu Leu Pro Gly Leu Arg Leu Gly 85
90 95Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val
Val Thr Met Lys Ser 100 105
110Ser Leu Met Phe Leu Ala Lys Val Gly Ser Gln Ser Ile Ala Ala Tyr
115 120 125Cys Asn Tyr Thr Gln Tyr Gln
Pro Arg Val Leu Ala Val Ile Gly Pro 130 135
140His Ser Ser Glu Leu Ala Leu Ile Thr Gly Lys Phe Phe Ser Phe
Phe145 150 155 160Leu Met
Pro Gln Val Ser Tyr Ser Ala Ser Met Asp Arg Leu Ser Asp
165 170 175Arg Glu Thr Phe Pro Ser Phe
Phe Arg Thr Val Pro Ser Asp Arg Val 180 185
190Gln Leu Gln Ala Val Val Thr Leu Leu Gln Asn Phe Ser Trp
Asn Trp 195 200 205Val Ala Ala Leu
Gly Ser Asp Asp Asp Tyr Gly Arg Glu Gly Leu Ser 210
215 220Ile Phe Ser Ser Leu Ala Asn Ala Arg Gly Ile Cys
Ile Ala His Glu225 230 235
240Gly Leu Val Pro Gln His Asp Thr Ser Gly Gln Gln Leu Gly Lys Val
245 250 255Leu Asp Val Leu Arg
Gln Val Asn Gln Ser Lys Val Gln Val Val Val 260
265 270Leu Phe Ala Ser Ala Arg Ala Val Tyr Ser Leu Phe
Ser Tyr Ser Ile 275 280 285His His
Gly Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ser Trp Leu 290
295 300Thr Ser Asp Leu Val Met Thr Leu Pro Asn Ile
Ala Arg Val Gly Thr305 310 315
320Val Leu Gly Phe Leu Gln Arg Gly Ala Leu Leu Pro Glu Phe Ser His
325 330 335Tyr Val Glu Thr
His Leu Ala Leu Ala Ala Asp Pro Ala Phe Cys Ala 340
345 350Ser Leu Asn Ala Glu Leu Asp Leu Glu Glu His
Val Met Gly Gln Arg 355 360 365Cys
Pro Arg Cys Asp Asp Ile Met Leu Gln Asn Leu Ser Ser Gly Leu 370
375 380Leu Gln Asn Leu Ser Ala Gly Gln Leu His
His Gln Ile Phe Ala Thr385 390 395
400Tyr Ala Ala Val Tyr Ser Val Ala Gln Ala Leu His Asn Thr Leu
Gln 405 410 415Cys Asn Val
Ser His Cys His Val Ser Glu His Val Leu Pro Trp Gln 420
425 430Leu Leu Glu Asn Met Tyr Asn Met Ser Phe
His Ala Arg Asp Leu Thr 435 440
445Leu Gln Phe Asp Ala Glu Gly Asn Val Asp Met Glu Tyr Asp Leu Lys 450
455 460Met Trp Val Trp Gln Ser Pro Thr
Pro Val Leu His Thr Val Gly Thr465 470
475 480Phe Asn Gly Thr Leu Gln Leu Gln Gln Ser Lys Met
Tyr Trp Pro Gly 485 490
495Asn Gln Val Pro Val Ser Gln Cys Ser Arg Gln Cys Lys Asp Gly Gln
500 505 510Val Arg Arg Val Lys Gly
Phe His Ser Cys Cys Tyr Asp Cys Val Asp 515 520
525Cys Lys Ala Gly Ser Tyr Arg Lys His Pro Asp Asp Phe Thr
Cys Thr 530 535 540Pro Cys Asn Gln Asp
Gln Trp Ser Pro Glu Lys Ser Thr Ala Cys Leu545 550
555 560Pro Arg Arg Pro Lys Phe Leu Ala Trp Gly
Glu Pro Val Val Leu Ser 565 570
575Leu Leu Leu Leu Leu Cys Leu Val Leu Gly Leu Ala Leu Ala Ala Leu
580 585 590Gly Leu Ser Val His
His Trp Asp Ser Pro Leu Val Gln Ala Ser Gly 595
600 605Gly Ser Gln Phe Cys Phe Gly Leu Ile Cys Leu Gly
Leu Phe Cys Leu 610 615 620Ser Val Leu
Leu Phe Pro Gly Arg Pro Ser Ser Ala Ser Cys Leu Ala625
630 635 640Gln Gln Pro Met Ala His Leu
Pro Leu Thr Gly Cys Leu Ser Thr Leu 645
650 655Phe Leu Gln Ala Ala Glu Thr Phe Val Glu Ser Glu
Leu Pro Leu Ser 660 665 670Trp
Ala Asn Trp Leu Cys Ser Tyr Leu Arg Gly Leu Trp Ala Trp Leu 675
680 685Val Val Leu Leu Ala Thr Phe Val Glu
Ala Ala Leu Cys Ala Trp Tyr 690 695
700Leu Ile Ala Phe Pro Pro Glu Val Val Thr Asp Trp Ser Val Leu Pro705
710 715 720Thr Glu Val Leu
Glu His Cys His Val Arg Ser Trp Val Ser Leu Gly 725
730 735Leu Val His Ile Thr Asn Ala Met Leu Ala
Phe Leu Cys Phe Leu Gly 740 745
750Thr Phe Leu Val Gln Ser Gln Pro Gly Arg Tyr Asn Arg Ala Arg Gly
755 760 765Leu Thr Phe Ala Met Leu Ala
Tyr Phe Ile Thr Trp Val Ser Phe Val 770 775
780Pro Leu Leu Ala Asn Val Gln Val Ala Tyr Gln Pro Ala Val Gln
Met785 790 795 800Gly Ala
Ile Leu Val Cys Ala Leu Gly Ile Leu Val Thr Phe His Leu
805 810 815Pro Lys Cys Tyr Val Leu Leu
Trp Leu Pro Lys Leu Asn Thr Gln Glu 820 825
830Phe Phe Leu Gly Arg Asn Ala Lys Lys Ala Ala Asp Glu Asn
Ser Gly 835 840 845Gly Gly Glu Ala
Ala Gln Gly His Asn Glu 850 85528858PRTMus musculus
28Met Pro Ala Leu Ala Ile Met Gly Leu Ser Leu Ala Ala Phe Leu Glu1
5 10 15Leu Gly Met Gly Ala Ser
Leu Cys Leu Ser Gln Gln Phe Lys Ala Gln 20 25
30Gly Asp Tyr Ile Leu Gly Gly Leu Phe Pro Leu Gly Ser
Thr Glu Glu 35 40 45Ala Thr Leu
Asn Gln Arg Thr Gln Pro Asn Ser Ile Pro Cys Asn Arg 50
55 60Phe Ser Pro Leu Gly Leu Phe Leu Ala Met Ala Met
Lys Met Ala Val65 70 75
80Glu Glu Ile Asn Asn Gly Ser Ala Leu Leu Pro Gly Leu Arg Leu Gly
85 90 95Tyr Asp Leu Phe Asp Thr
Cys Ser Glu Pro Val Val Thr Met Lys Ser 100
105 110Ser Leu Met Phe Leu Ala Lys Val Gly Ser Gln Ser
Ile Ala Ala Tyr 115 120 125Cys Asn
Tyr Thr Gln Tyr Gln Pro Arg Val Leu Ala Val Ile Gly Pro 130
135 140His Ser Ser Glu Leu Ala Leu Ile Thr Gly Lys
Phe Phe Ser Phe Phe145 150 155
160Leu Met Pro Gln Val Ser Tyr Ser Ala Ser Met Asp Arg Leu Ser Asp
165 170 175Arg Glu Thr Phe
Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val 180
185 190Gln Leu Gln Ala Val Val Thr Leu Leu Gln Asn
Phe Ser Trp Asn Trp 195 200 205Val
Ala Ala Leu Gly Ser Asp Asp Asp Tyr Gly Arg Glu Gly Leu Ser 210
215 220Ile Phe Ser Ser Leu Ala Asn Ala Arg Gly
Ile Cys Ile Ala His Glu225 230 235
240Gly Leu Val Pro Gln His Asp Thr Ser Gly Gln Gln Leu Gly Lys
Val 245 250 255Leu Asp Val
Leu Arg Gln Val Asn Gln Ser Lys Val Gln Val Val Val 260
265 270Leu Phe Ala Ser Ala Arg Ala Val Tyr Ser
Leu Phe Ser Tyr Ser Ile 275 280
285His His Gly Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ser Trp Leu 290
295 300Thr Ser Asp Leu Val Met Thr Leu
Pro Asn Ile Ala Arg Val Gly Thr305 310
315 320Val Leu Gly Phe Leu Gln Arg Gly Ala Leu Leu Pro
Glu Phe Ser His 325 330
335Tyr Val Glu Thr His Leu Ala Leu Ala Ala Asp Pro Ala Phe Cys Ala
340 345 350Ser Leu Asn Ala Glu Leu
Asp Leu Glu Glu His Val Met Gly Gln Arg 355 360
365Cys Pro Arg Cys Asp Asp Ile Met Leu Gln Asn Leu Ser Ser
Gly Leu 370 375 380Leu Gln Asn Leu Ser
Ala Gly Gln Leu His His Gln Ile Phe Ala Thr385 390
395 400Tyr Ala Ala Val Tyr Ser Val Ala Gln Ala
Leu His Asn Thr Leu Gln 405 410
415Cys Asn Val Ser His Cys His Val Ser Glu His Val Leu Pro Trp Gln
420 425 430Leu Leu Glu Asn Met
Tyr Asn Met Ser Phe His Ala Arg Asp Leu Thr 435
440 445Leu Gln Phe Asp Ala Glu Gly Asn Val Asp Met Glu
Tyr Asp Leu Lys 450 455 460Met Trp Val
Trp Gln Ser Pro Thr Pro Val Leu His Thr Val Gly Thr465
470 475 480Phe Asn Gly Thr Leu Gln Leu
Gln Gln Ser Lys Met Tyr Trp Pro Gly 485
490 495Asn Gln Val Pro Val Ser Gln Cys Ser Arg Gln Cys
Lys Asp Gly Gln 500 505 510Val
Arg Arg Val Lys Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp 515
520 525Cys Lys Ala Gly Ser Tyr Arg Lys His
Pro Asp Asp Phe Thr Cys Thr 530 535
540Pro Cys Asn Gln Asp Gln Trp Ser Pro Glu Lys Ser Thr Ala Cys Leu545
550 555 560Pro Arg Arg Pro
Lys Phe Leu Ala Trp Gly Glu Pro Val Val Leu Ser 565
570 575Leu Leu Leu Leu Leu Cys Leu Val Leu Gly
Leu Ala Leu Ala Ala Leu 580 585
590Gly Leu Ser Val His His Trp Asp Ser Pro Leu Val Gln Ala Ser Gly
595 600 605Gly Ser Gln Phe Cys Phe Gly
Leu Ile Cys Leu Gly Leu Phe Cys Leu 610 615
620Ser Val Leu Leu Phe Pro Gly Arg Pro Ser Ser Ala Ser Cys Leu
Ala625 630 635 640Gln Gln
Pro Met Ala His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu
645 650 655Phe Leu Gln Ala Ala Glu Thr
Phe Val Glu Ser Glu Leu Pro Leu Ser 660 665
670Trp Ala Asn Trp Leu Cys Ser Tyr Leu Arg Gly Leu Trp Ala
Trp Leu 675 680 685Val Val Leu Leu
Ala Thr Phe Val Glu Ala Ala Leu Cys Ala Trp Tyr 690
695 700Leu Ile Ala Phe Pro Pro Glu Val Val Thr Asp Trp
Ser Val Leu Pro705 710 715
720Thr Glu Val Leu Glu His Cys His Val Arg Ser Trp Val Ser Leu Gly
725 730 735Leu Val His Ile Thr
Asn Ala Met Leu Ala Phe Leu Cys Phe Leu Gly 740
745 750Thr Phe Leu Val Gln Ser Gln Pro Gly Arg Tyr Asn
Arg Ala Arg Gly 755 760 765Leu Thr
Phe Ala Met Leu Ala Tyr Phe Ile Thr Trp Val Ser Phe Val 770
775 780Pro Leu Leu Ala Asn Val Gln Val Ala Tyr Gln
Pro Ala Val Gln Met785 790 795
800Gly Ala Ile Leu Val Cys Ala Leu Gly Ile Leu Val Thr Phe His Leu
805 810 815Pro Lys Cys Tyr
Val Leu Leu Trp Leu Pro Lys Leu Asn Thr Gln Glu 820
825 830Phe Phe Leu Gly Arg Asn Ala Lys Lys Ala Ala
Asp Glu Asn Ser Gly 835 840 845Gly
Gly Glu Ala Ala Gln Gly His Asn Glu 850 85529858PRTMus
musculus 29Met Pro Ala Leu Ala Ile Met Gly Leu Ser Leu Ala Ala Phe Leu
Glu1 5 10 15Leu Gly Met
Gly Ala Ser Leu Cys Leu Ser Gln Gln Phe Lys Ala Gln 20
25 30Gly Asp Tyr Ile Leu Gly Gly Leu Phe Pro
Leu Gly Ser Thr Glu Glu 35 40
45Ala Thr Leu Asn Gln Arg Thr Gln Pro Asn Ser Ile Pro Cys Asn Arg 50
55 60Phe Ser Pro Leu Gly Leu Phe Leu Ala
Met Ala Met Lys Met Ala Val65 70 75
80Glu Glu Ile Asn Asn Gly Ser Ala Leu Leu Pro Gly Leu Arg
Leu Gly 85 90 95Tyr Asp
Leu Phe Asp Thr Cys Ser Glu Pro Val Val Thr Met Lys Ser 100
105 110Ser Leu Met Phe Leu Ala Lys Val Gly
Ser Gln Ser Ile Ala Ala Tyr 115 120
125Cys Asn Tyr Thr Gln Tyr Gln Pro Arg Val Leu Ala Val Ile Gly Pro
130 135 140His Ser Ser Glu Leu Ala Leu
Ile Thr Gly Lys Phe Phe Ser Phe Phe145 150
155 160Leu Met Pro Gln Val Ser Tyr Ser Ala Ser Met Asp
Arg Leu Ser Asp 165 170
175Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val
180 185 190Gln Leu Gln Ala Val Val
Thr Leu Leu Gln Asn Phe Ser Trp Asn Trp 195 200
205Val Ala Ala Leu Gly Ser Asp Asp Asp Tyr Gly Arg Glu Gly
Leu Ser 210 215 220Ile Phe Ser Ser Leu
Ala Asn Ala Arg Gly Ile Cys Ile Ala His Glu225 230
235 240Gly Leu Val Pro Gln His Asp Thr Ser Gly
Gln Gln Leu Gly Lys Val 245 250
255Leu Asp Val Leu Arg Gln Val Asn Gln Ser Lys Val Gln Val Val Val
260 265 270Leu Phe Ala Ser Ala
Arg Ala Val Tyr Ser Leu Phe Ser Tyr Ser Ile 275
280 285His His Gly Leu Ser Pro Lys Val Trp Val Ala Ser
Glu Ser Trp Leu 290 295 300Thr Ser Asp
Leu Val Met Thr Leu Pro Asn Ile Ala Arg Val Gly Thr305
310 315 320Val Leu Gly Phe Leu Gln Arg
Gly Ala Leu Leu Pro Glu Phe Ser His 325
330 335Tyr Val Glu Thr His Leu Ala Leu Ala Ala Asp Pro
Ala Phe Cys Ala 340 345 350Ser
Leu Asn Ala Glu Leu Asp Leu Glu Glu His Val Met Gly Gln Arg 355
360 365Cys Pro Arg Cys Asp Asp Ile Met Leu
Gln Asn Leu Ser Ser Gly Leu 370 375
380Leu Gln Asn Leu Ser Ala Gly Gln Leu His His Gln Ile Phe Ala Thr385
390 395 400Tyr Ala Ala Val
Tyr Ser Val Ala Gln Ala Leu His Asn Thr Leu Gln 405
410 415Cys Asn Val Ser His Cys His Val Ser Glu
His Val Leu Pro Trp Gln 420 425
430Leu Leu Glu Asn Met Tyr Asn Met Ser Phe His Ala Arg Asp Leu Thr
435 440 445Leu Gln Phe Asp Ala Glu Gly
Asn Val Asp Met Glu Tyr Asp Leu Lys 450 455
460Met Trp Val Trp Gln Ser Pro Thr Pro Val Leu His Thr Val Gly
Thr465 470 475 480Phe Asn
Gly Thr Leu Gln Leu Gln Gln Ser Lys Met Tyr Trp Pro Gly
485 490 495Asn Gln Val Pro Val Ser Gln
Cys Ser Arg Gln Cys Lys Asp Gly Gln 500 505
510Val Arg Arg Val Lys Gly Phe His Ser Cys Cys Tyr Asp Cys
Val Asp 515 520 525Cys Lys Ala Gly
Ser Tyr Arg Lys His Pro Asp Asp Phe Thr Cys Thr 530
535 540Pro Cys Asn Gln Asp Gln Trp Ser Pro Glu Lys Ser
Thr Ala Cys Leu545 550 555
560Pro Arg Arg Pro Lys Phe Leu Ala Trp Gly Glu Pro Val Val Leu Ser
565 570 575Leu Leu Leu Leu Leu
Cys Leu Val Leu Gly Leu Ala Leu Ala Ala Leu 580
585 590Gly Leu Ser Val His His Trp Asp Ser Pro Leu Val
Gln Ala Ser Gly 595 600 605Gly Ser
Gln Phe Cys Phe Gly Leu Ile Cys Leu Gly Leu Phe Cys Leu 610
615 620Ser Val Leu Leu Phe Pro Gly Arg Pro Ser Ser
Ala Ser Cys Leu Ala625 630 635
640Gln Gln Pro Met Ala His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu
645 650 655Phe Leu Gln Ala
Ala Glu Thr Phe Val Glu Ser Glu Leu Pro Leu Ser 660
665 670Trp Ala Asn Trp Leu Cys Ser Tyr Leu Arg Gly
Leu Trp Ala Trp Leu 675 680 685Val
Val Leu Leu Ala Thr Phe Val Glu Ala Ala Leu Cys Ala Trp Tyr 690
695 700Leu Ile Ala Phe Pro Pro Glu Val Val Thr
Asp Trp Ser Val Leu Pro705 710 715
720Thr Glu Val Leu Glu His Cys His Val Arg Ser Trp Val Ser Leu
Gly 725 730 735Leu Val His
Ile Thr Asn Ala Met Leu Ala Phe Leu Cys Phe Leu Gly 740
745 750Thr Phe Leu Val Gln Ser Gln Pro Gly Arg
Tyr Asn Arg Ala Arg Gly 755 760
765Leu Thr Phe Ala Met Leu Ala Tyr Phe Ile Thr Trp Val Ser Phe Val 770
775 780Pro Leu Leu Ala Asn Val Gln Val
Ala Tyr Gln Pro Ala Val Gln Met785 790
795 800Gly Ala Ile Leu Val Cys Ala Leu Gly Ile Leu Val
Thr Phe His Leu 805 810
815Pro Lys Cys Tyr Val Leu Leu Trp Leu Pro Lys Leu Asn Thr Gln Glu
820 825 830Phe Phe Leu Gly Arg Asn
Ala Lys Lys Ala Ala Asp Glu Asn Ser Gly 835 840
845Gly Gly Glu Ala Ala Gln Gly His Asn Glu 850
85530858PRTMus musculus 30Met Pro Ala Leu Ala Ile Met Gly Leu Ser
Leu Ala Ala Phe Leu Glu1 5 10
15Leu Gly Met Gly Ala Ser Leu Cys Leu Ser Gln Gln Phe Lys Ala Gln
20 25 30Gly Asp Tyr Ile Leu Gly
Gly Leu Phe Pro Leu Gly Ser Thr Glu Glu 35 40
45Ala Thr Leu Asn Gln Arg Thr Gln Pro Asn Ser Ile Pro Cys
Asn Arg 50 55 60Phe Ser Pro Leu Gly
Leu Phe Leu Ala Met Ala Met Lys Met Ala Val65 70
75 80Glu Glu Ile Asn Asn Gly Ser Ala Leu Leu
Pro Gly Leu Arg Leu Gly 85 90
95Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Thr Met Lys Ser
100 105 110Ser Leu Met Phe Leu
Ala Lys Val Gly Ser Gln Ser Ile Ala Ala Tyr 115
120 125Cys Asn Tyr Thr Gln Tyr Gln Pro Arg Val Leu Ala
Val Ile Gly Pro 130 135 140His Ser Ser
Glu Leu Ala Leu Ile Thr Gly Lys Phe Phe Ser Phe Phe145
150 155 160Leu Met Pro Gln Val Ser Tyr
Ser Ala Ser Met Asp Arg Leu Ser Asp 165
170 175Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro
Ser Asp Arg Val 180 185 190Gln
Leu Gln Ala Val Val Thr Leu Leu Gln Asn Phe Ser Trp Asn Trp 195
200 205Val Ala Ala Leu Gly Ser Asp Asp Asp
Tyr Gly Arg Glu Gly Leu Ser 210 215
220Ile Phe Ser Ser Leu Ala Asn Ala Arg Gly Ile Cys Ile Ala His Glu225
230 235 240Gly Leu Val Pro
Gln His Asp Thr Ser Gly Gln Gln Leu Gly Lys Val 245
250 255Leu Asp Val Leu Arg Gln Val Asn Gln Ser
Lys Val Gln Val Val Val 260 265
270Leu Phe Ala Ser Ala Arg Ala Val Tyr Ser Leu Phe Ser Tyr Ser Ile
275 280 285His His Gly Leu Ser Pro Lys
Val Trp Val Ala Ser Glu Ser Trp Leu 290 295
300Thr Ser Asp Leu Val Met Thr Leu Pro Asn Ile Ala Arg Val Gly
Thr305 310 315 320Val Leu
Gly Phe Leu Gln Arg Gly Ala Leu Leu Pro Glu Phe Ser His
325 330 335Tyr Val Glu Thr His Leu Ala
Leu Ala Ala Asp Pro Ala Phe Cys Ala 340 345
350Ser Leu Asn Ala Glu Leu Asp Leu Glu Glu His Val Met Gly
Gln Arg 355 360 365Cys Pro Arg Cys
Asp Asp Ile Met Leu Gln Asn Leu Ser Ser Gly Leu 370
375 380Leu Gln Asn Leu Ser Ala Gly Gln Leu His His Gln
Ile Phe Ala Thr385 390 395
400Tyr Ala Ala Val Tyr Ser Val Ala Gln Ala Leu His Asn Thr Leu Gln
405 410 415Cys Asn Val Ser His
Cys His Val Ser Glu His Val Leu Pro Trp Gln 420
425 430Leu Leu Glu Asn Met Tyr Asn Met Ser Phe His Ala
Arg Asp Leu Thr 435 440 445Leu Gln
Phe Asp Ala Glu Gly Asn Val Asp Met Glu Tyr Asp Leu Lys 450
455 460Met Trp Val Trp Gln Ser Pro Thr Pro Val Leu
His Thr Val Gly Thr465 470 475
480Phe Asn Gly Thr Leu Gln Leu Gln Gln Ser Lys Met Tyr Trp Pro Gly
485 490 495Asn Gln Val Pro
Val Ser Gln Cys Ser Arg Gln Cys Lys Asp Gly Gln 500
505 510Val Arg Arg Val Lys Gly Phe His Ser Cys Cys
Tyr Asp Cys Val Asp 515 520 525Cys
Lys Ala Gly Ser Tyr Arg Lys His Pro Asp Asp Phe Thr Cys Thr 530
535 540Pro Cys Asn Gln Asp Gln Trp Ser Pro Glu
Lys Ser Thr Ala Cys Leu545 550 555
560Pro Arg Arg Pro Lys Phe Leu Ala Trp Gly Glu Pro Val Val Leu
Ser 565 570 575Leu Leu Leu
Leu Leu Cys Leu Val Leu Gly Leu Ala Leu Ala Ala Leu 580
585 590Gly Leu Ser Val His His Trp Asp Ser Pro
Leu Val Gln Ala Ser Gly 595 600
605Gly Ser Gln Phe Cys Phe Gly Leu Ile Cys Leu Gly Leu Phe Cys Leu 610
615 620Ser Val Leu Leu Phe Pro Gly Arg
Pro Ser Ser Ala Ser Cys Leu Ala625 630
635 640Gln Gln Pro Met Ala His Leu Pro Leu Thr Gly Cys
Leu Ser Thr Leu 645 650
655Phe Leu Gln Ala Ala Glu Thr Phe Val Glu Ser Glu Leu Pro Leu Ser
660 665 670Trp Ala Asn Trp Leu Cys
Ser Tyr Leu Arg Gly Leu Trp Ala Trp Leu 675 680
685Val Val Leu Leu Ala Thr Phe Val Glu Ala Ala Leu Cys Ala
Trp Tyr 690 695 700Leu Ile Ala Phe Pro
Pro Glu Val Val Thr Asp Trp Ser Val Leu Pro705 710
715 720Thr Glu Val Leu Glu His Cys His Val Arg
Ser Trp Val Ser Leu Gly 725 730
735Leu Val His Ile Thr Asn Ala Met Leu Ala Phe Leu Cys Phe Leu Gly
740 745 750Thr Phe Leu Val Gln
Ser Gln Pro Gly Arg Tyr Asn Arg Ala Arg Gly 755
760 765Leu Thr Phe Ala Met Leu Ala Tyr Phe Ile Thr Trp
Val Ser Phe Val 770 775 780Pro Leu Leu
Ala Asn Val Gln Val Ala Tyr Gln Pro Ala Val Gln Met785
790 795 800Gly Ala Ile Leu Val Cys Ala
Leu Gly Ile Leu Val Thr Phe His Leu 805
810 815Pro Lys Cys Tyr Val Leu Leu Trp Leu Pro Lys Leu
Asn Thr Gln Glu 820 825 830Phe
Phe Leu Gly Arg Asn Ala Lys Lys Ala Ala Asp Glu Asn Ser Gly 835
840 845Gly Gly Glu Ala Ala Gln Glu His Asn
Glu 850 85531858PRTMus musculus 31Met Pro Ala Leu Ala
Ile Met Gly Leu Ser Leu Ala Ala Phe Leu Glu1 5
10 15Leu Gly Met Gly Ala Ser Leu Cys Leu Ser Gln
Gln Phe Lys Ala Gln 20 25
30Gly Asp Tyr Ile Leu Gly Gly Leu Phe Pro Leu Gly Ser Thr Glu Glu
35 40 45Ala Thr Leu Asn Gln Arg Ala Gln
Pro Asn Ser Thr Leu Cys Asn Arg 50 55
60Phe Ser Pro Leu Gly Leu Phe Leu Ala Met Ala Met Lys Met Ala Val65
70 75 80Glu Glu Ile Asn Asn
Gly Ser Ala Leu Leu Pro Gly Leu Arg Leu Gly 85
90 95Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val
Val Thr Met Lys Ser 100 105
110Ser Leu Met Phe Leu Ala Lys Val Gly Ser Gln Ser Ile Ala Ala Tyr
115 120 125Cys Asn Tyr Thr Gln Tyr Gln
Pro Arg Val Leu Ala Val Ile Gly Pro 130 135
140His Ser Ser Glu Leu Ala Leu Ile Thr Gly Lys Phe Phe Ser Phe
Phe145 150 155 160Leu Met
Pro Gln Val Ser Tyr Ser Ala Ser Met Asp Arg Leu Ser Asp
165 170 175Arg Glu Thr Phe Pro Ser Phe
Phe Arg Thr Val Pro Ser Asp Arg Val 180 185
190Gln Leu Gln Ala Val Val Thr Leu Leu Gln Asn Phe Ser Trp
Asn Trp 195 200 205Val Ala Ala Leu
Gly Ser Asp Asp Asp Tyr Gly Arg Glu Gly Leu Ser 210
215 220Ile Phe Ser Ser Leu Ala Asn Ala Arg Gly Ile Cys
Ile Ala His Glu225 230 235
240Gly Leu Val Pro Gln His Asp Thr Ser Gly Gln Gln Leu Gly Lys Val
245 250 255Leu Asp Val Leu Arg
Gln Val Asn Gln Ser Lys Val Gln Val Val Val 260
265 270Leu Phe Ala Ser Ala Arg Ala Val Tyr Ser Leu Phe
Ser Tyr Ser Ile 275 280 285His His
Gly Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ser Trp Leu 290
295 300Thr Ser Asp Leu Val Met Thr Leu Pro Asn Ile
Ala Arg Val Gly Thr305 310 315
320Val Leu Gly Phe Leu Gln Arg Gly Ala Leu Leu Pro Glu Phe Ser His
325 330 335Tyr Val Glu Thr
His Leu Ala Leu Ala Ala Asp Pro Ala Phe Cys Ala 340
345 350Ser Leu Asn Ala Glu Leu Asp Leu Glu Glu His
Val Met Gly Gln Arg 355 360 365Cys
Pro Gln Cys Asp Asp Ile Met Leu Gln Asn Leu Ser Ser Gly Leu 370
375 380Leu Gln Asn Leu Ser Ala Gly Gln Leu His
His Gln Ile Phe Ala Thr385 390 395
400Tyr Ala Ala Val Tyr Ser Val Ala Gln Ala Leu His Asn Thr Leu
Gln 405 410 415Cys Asn Val
Ser His Cys His Val Ser Glu His Val Leu Pro Trp Gln 420
425 430Leu Leu Glu Asn Met Tyr Asn Met Ser Phe
His Ala Arg Asp Leu Thr 435 440
445Leu Gln Phe Asp Ala Glu Gly Asn Val Asp Met Glu Tyr Asp Leu Lys 450
455 460Met Trp Val Trp Gln Ser Pro Thr
Pro Val Leu His Thr Val Gly Thr465 470
475 480Phe Asn Gly Thr Leu Gln Leu Gln Gln Ser Lys Met
Tyr Trp Pro Gly 485 490
495Asn Gln Val Pro Val Ser Gln Cys Ser Arg Gln Cys Lys Asp Gly Gln
500 505 510Val Arg Arg Val Lys Gly
Phe His Ser Cys Cys Tyr Asp Cys Val Asp 515 520
525Cys Lys Ala Gly Ser Tyr Arg Lys His Pro Asp Asp Phe Thr
Cys Thr 530 535 540Pro Cys Asn Gln Asp
Gln Trp Ser Pro Glu Lys Ser Thr Ala Cys Leu545 550
555 560Pro Arg Arg Pro Lys Phe Leu Ala Trp Gly
Glu Pro Val Val Leu Ser 565 570
575Leu Leu Leu Leu Leu Cys Leu Val Leu Gly Leu Ala Leu Ala Ala Leu
580 585 590Gly Leu Ser Val His
His Trp Asp Ser Pro Leu Val Gln Ala Ser Gly 595
600 605Gly Ser Gln Phe Cys Phe Gly Leu Ile Cys Leu Gly
Leu Phe Cys Leu 610 615 620Ser Val Leu
Leu Phe Pro Gly Arg Pro Ser Ser Ala Ser Cys Leu Ala625
630 635 640Gln Gln Pro Met Ala His Leu
Pro Leu Thr Gly Cys Leu Ser Thr Leu 645
650 655Phe Leu Gln Ala Ala Glu Thr Phe Val Glu Ser Glu
Leu Pro Leu Ser 660 665 670Trp
Ala Asn Trp Leu Cys Ser Tyr Leu Arg Gly Leu Trp Ala Trp Leu 675
680 685Val Val Leu Leu Ala Thr Phe Val Glu
Ala Ala Leu Cys Ala Trp Tyr 690 695
700Leu Thr Ala Phe Pro Pro Glu Val Val Thr Asp Trp Ser Val Leu Pro705
710 715 720Thr Glu Val Leu
Glu His Cys His Val Arg Ser Trp Val Ser Leu Gly 725
730 735Leu Val His Ile Thr Asn Ala Met Leu Ala
Phe Leu Cys Phe Leu Gly 740 745
750Thr Phe Leu Val Gln Ser Gln Pro Gly Arg Tyr Asn Arg Ala Arg Gly
755 760 765Leu Thr Phe Ala Met Leu Ala
Tyr Phe Ile Thr Trp Val Ser Phe Val 770 775
780Pro Leu Leu Ala Asn Val Gln Val Ala Tyr Gln Pro Ala Val Gln
Met785 790 795 800Gly Ala
Ile Leu Val Cys Ala Leu Gly Ile Leu Val Thr Phe His Leu
805 810 815Pro Lys Cys Tyr Val Leu Leu
Trp Leu Pro Lys Leu Asn Thr Gln Glu 820 825
830Phe Phe Leu Gly Arg Asn Ala Lys Lys Ala Ala Asp Glu Asn
Ser Gly 835 840 845Gly Gly Glu Ala
Ala Gln Glu His Asn Glu 850 855
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